Project description:Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we hypothesized that a set of genes co-evolved with BGCs to support biosynthetic proficiency must exist in those strains, and that their identification may provide universal strategies to improve the productivity of other strains. We show here that genes co-evolved with natural product BGCs in Streptomyces can be identified by phylogenomics analysis. Among the 597 genes that co-evolved with polyketide BGCs, 11 genes in the “coenzyme” category have been examined, including a gene cluster encoding for the co-factor pyrroloquinoline quinone (PQQ). When the pqq gene cluster was engineered into 11 Streptomyces strains, it enhanced production of 16,385 metabolites, including 36 known natural products with up to 40-fold improvement and several activated silent gene clusters. This study provides a new engineering strategy for improving polyketide production and discovering new biosynthetic gene clusters.
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:Biofilms are ubiquitous in natural, medical, and engineering environments. While most antibiotics that primarily aim to inhibit cell growth may result in bacterial drug resistance, biofilm inhibitors do not affect cell growth and there is less chance of developing resistance. This work sought to identify novel, non-toxic and potent biofilm inhibitors from Streptomyces bacteria for reducing the biofilm formation of Pseudomonas aeruginosa PAO1. Out of 4300 Streptomyces strains, one species produced and secreted peptide(s) to inhibit P. aeruginosa biofilm formation by 93% without affecting the growth of planktonic cells. Global transcriptome analyses (DNA microarray) revealed that the supernatant of the Streptomyces 230 strain induced phenazine, pyoverdine, and pyochelin synthesis genes. Electron microscopy showed that the supernatant of Streptomyces 230 strain reduced the production of polymeric matrix in P. aeruginosa biofilm cells, while the Streptomyces species enhanced swarming motility of P. aeruginosa. Therefore, current study suggests that Streptomyces bacteria are an important resource of biofilm inhibitors as well as antibiotics.
Project description:Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we hypothesized that a set of genes co-evolved with BGCs to support biosynthetic proficiency must exist in those strains, and that their identification may provide universal strategies to improve the productivity of other strains. We show here that genes co-evolved with natural product BGCs in Streptomyces can be identified by phylogenomics analysis. Among the 597 genes that co-evolved with polyketide BGCs, 11 genes in the “coenzyme” category have been examined, including a gene cluster encoding for the co-factor pyrroloquinoline quinone (PQQ). When the pqq gene cluster was engineered into 11 Streptomyces strains, it enhanced production of 16,385 metabolites, including 36 known natural products with up to 40-fold improvement and several activated silent gene clusters. This study provides a new engineering strategy for improving polyketide production and discovering new biosynthetic gene clusters.
Project description:We report the mRNA and small RNA transcriptomes of Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces venezuelae. We identified dozens of new conserved sRNAs and antisense RNAs, including a prominent group of antisense RNAs termed ‘cutoRNAs’ that result from overlap of the 3′ ends of convergently transcribed mRNAs. In addition, we observed abundant unique ncRNAs, including many within secondary metabolic gene clusters. For each of the three species (S. coelicolor, S. avermitilis, S. venezuelae) two libraries were created. The first was a long-read library, where total RNA was depleted of rRNA. The second was a short read library, where total RNA was size selected (40-300 nucleotides) prior to library creation. The long-read library allowed the profiling of mRNAs and asRNAs, while the short-read library was enriched for small RNAs (sRNAs).
Project description:We report the mRNA and small RNA transcriptomes of Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces venezuelae. We identified dozens of new conserved sRNAs and antisense RNAs, including a prominent group of antisense RNAs termed ‘cutoRNAs’ that result from overlap of the 3′ ends of convergently transcribed mRNAs. In addition, we observed abundant unique ncRNAs, including many within secondary metabolic gene clusters.
Project description:SigE is a sigma factor found in Streptomyces species and is the key regulator of cell envelope stress response in Streptomyces coelicolor. This ChIP-Seq experiment was carried out to determine the binding sites of SigE under conditions of cell envelope stress induced by 10 ug / ml vancomycin for 30 minutes. A 3xFLAG tagged SigE was introduced in a SigE deletion strain and ChIP-Seq was carried out using M2 (Sigma Aldrich A2220) gel suspension. Non-immunoprecipitated (total) DNA preparations were used for background determination and the wild type M600 was used as the negative control.
Project description:The whiH gene is required for the differentiation of aerial hyphae into spores in Streptomyces species. It is a predicted member of the GntR family of transcription factors and has been shown to bind specifically to a sequence in its own promoter. This ChIP-Seq experiment was carried out to determine all the binding sites whiH binds to in the genome of Streptomyces venezuelae. A whiH deletion strain was made and a FLAG tagged whiH protein was expressed in it from a genome-integrated plasmid. Then anti-FLAG antibodies were used for chromatin immunoprecipitation followed by high throughput sequencing. The wild type Streptomyces venezuelae strain (ATCC 10712) was used as a negative control. For both the FLAG-WhiH strain and the WT strain, non-immunoprecipitated (total) DNA was also sequenced to arrive at a background enrichment which could be subtracted from the enrichment in the immunoprecipated sample.
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:We isolated and sequenced mRNA from Streptomyces venezuelae grown on solid medium that promotes exploratory behaviour in this bacterial species. The data was analyzed using DeSeq2 to identify genes that undergo changes in expression over time.