Project description:This study compared the genome of Streptomyces rimosus rimosus against that of Streptomyces coelicolor. It also compared 4 strains with changes in oxytetracycline production and derived from G7, the type strain, against G7. Keywords: Comparative genomic hybridization
Project description:We identified genome-wide binding regions of NdgR in Streptomyces coelicolor using chromatin immunoprecipitation sequencing (ChIP-seq). We constructed 6×myc-tagged NdgR strain using homologous recombination with myc-tagging vector. Analysis of the sequencing data aligned to Streptomyces coelicolor genome database (NC_003888).
Project description:We performed ribosome profiling which is the deep-sequencing of mRNA fragments protected by translating ribosome for two Streptomyces species through different growth phases to provide the translatome data
Project description:Two component sensor-response regulator systems (TCSs) are very common in the genomes of the Streptomyces species that have been fully sequenced to date. It has been suggested that this large number is an evolutionary response to the variable environment that Streptomyces encounter in soil. Notwithstanding this, TCSs are also more common in the sequenced genomes of other Actinomycetales when these are compared to the genomes of most other eubacteria. In this study, we have used DNA/DNA genome microarray analysis to compare fourteen Streptomyces species and one closely related genus to Streptomyces coelicolor in order to identify a core group of such systems. This core group is compared to the syntenous and non-syntenous TCSs present in the genome sequences of other Actinomycetales in order to separate the systems into those present in Actinomycetales in general, the Streptomyces specific systems and the species specific systems. Horizontal transfer does not seem to play a very important role in the evolution of the TCS complement analyzed in this study. However, cognate pairs do not necessarily seem to evolve at the same pace, which may indicate the evolutionary responses to environmental variation may be reflected differently in sequence changes within the two components of the TCSs. The overall analysis allowed subclassification of the orphan TCSs and the TCS cognate pairs and identification of possible targets for further study using gene knockouts, gene overexpression, reporter genes and yeast two hybrid analysis.
Project description:Toyocamycin is a member of the nucleoside antibiotic family and has been recognized as a promising fungicide for the control of plant diseases. However, low productivity of toyocamycin remains an important bottleneck in its industrial production. Therefore, dramatic improvements of strains for overproduction of toyocamycin are of great interest in applied microbiology research. In this study, we sequentially selected for mutations for multiple drug resistance to promote the overproduction of toyocamycin by Streptomyces diastatochromogenes 1628. The triple mutant strain, SD3145 (str str par), was obtained through sequential screenings. This strain showed an enhanced capacity to produce toyocamycin (1500 mg/L), 24-fold higher than the wild type in GYM liquid medium. This dramatic overproduction was attributed at least partially to the acquisition of an rsmG mutation and increased gene expression of toyA, which encodes a LuxR-family transcriptional regulator for toyocamycin biosynthesis. The expression of toyF and toyG, probably directly involved in toyocamycin biosynthesis, was also enhanced, contributing to toyocamycin overproduction. By addition of a small amount of scandium (ScCl3·6H2O), the mutant strain, SD3145, produced more toyocamycin (2664 mg/L) in TPM medium, which was the highest toyocamycin level produced in shake-flask fermentation by a streptomycete so far. We demonstrated that introduction of combined drug resistance mutations into S. diastatochromogenes 1628 resulted in an obvious increase in the toyocamycin production. The triple mutant strain, SD3145, generated in our study could be useful for improvement of industrial production of toyocamycin.
Project description:ppGpp is a ubiquitous small nucleotide messenger that mediates cellular self-protective responses under environmental stress. However, the mechanisms of ppGpp that control transcription and other metabolic processes depend on the species, and ppGpp regulates the same process via different mechanisms. The level of ppGpp is regulated by RelA/SpoT homolog (RSH) enzymes that synthesize and hydrolyze the alarmone. Here, we constructed a ppGpp0 strain and monitored the effects of ppGpp on the transcriptional level, physiology, and secondary metabiotic production in the antibiotic producer Streptomyces diastatochromogenes 1628. The results showed the cell division and growth of ppGpp0 increased by measurement of gene transcription and DCWs. The utilization of nitrogen was affected depending on the nitrogen type with a significantly higher DCW of the ppGpp0 mutant in the medium supplied with the yeast extract and a lower growth rate in the inorganic nitrogen ammonium salt. The ppGpp-mediated stringent response could not affect the usage of carbon resources. More importantly, ppGpp0 inhibited the expression of antibiotic clusters and the production of toyocamycin and tetramycin P. The antibiotic resistance was also significantly downregulated in the ppGpp0 mutant. In conclusion, this study showed detailed changes in ppGpp-mediated stringent responses on S. diastatochromogenes 1628 cell growth, nutrient utilization, morphological characteristics, antibiotic production, and resistance, which will provide insights into the role of ppGpp in Streptomyces. IMPORTANCE The ppGpp-mediated stringent response is widely distributed in Escherichia coli, Bacillus subtilis, Streptomyces, Staphylococcus aureus, etc. Stringent responses give strains the ability to resist environmental stresses, and survival from nutrition starvation, virulence, long-term persistence, biofilm formation, and gut colonization. ppGpp has many targets in cells and can reprogram DNA replication, transcription, ribosome biogenesis and function, and lipid metabolism. However, the mechanism of ppGpp to control transcription and other metabolic processes depends on the bacterial species and regulates the same process via a different mechanism. In Streptomyces, how ppGpp regulates the transcription remains to be elucidated. However, because ppGpp regulates many genes involved in primary and secondary metabolism, we compared the transcription and cell division, cell growth, morphological differentiation, antibiotic resistance, and secondary synthesis in the wild-type S. diastatochromogenes and ppGpp0 strains.