Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli.
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ABSTRACT:
SUBMITTER: Roberts AN
PROVIDER: S-EPMC1147873 | biostudies-other | 1987 Apr
REPOSITORIES: biostudies-other
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Project description:Streptomyces lividans is an attractive host for production of heterologous proteins and secondary metabolites of other Streptomyces species. To fully harness the industrial potential of S. lividans, understanding its metabolism and genetic regulatory elements is essential. This study aimed to determine its transcription unit (TU) architecture and elucidate its diverse regulatory elements, including promoters, ribosome binding sites, 5'-untranslated regions, and transcription terminators. Total 1,978 transcription start sites and 1,640 transcript 3'-end positions were identified, which were integrated to determine 1,300 TUs, consistent with transcriptomic profiles. The conserved promoter sequences were found as 5'-TANNNT and 5'-TGAC, representing the -10 and -35 elements, respectively. Analysis of transcript 3'-end positions revealed the presence of distinctive terminator sequences and the RNA stem structure responsible for the determination of the 3'-boundary of a transcript. Functionally related genes are likely to be regulated simultaneously by using similar promoters and being transcribed as a poly-cistronic TU. Poly-cistronic TUs were further processed or alternatively transcribed into multiple TUs to fine-regulate individual genes in response to environmental conditions. The TU information and regulatory elements identified will serve as invaluable resources for understanding the complex regulatory mechanisms of S. lividans and to elevate its industrial potential.
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Project description:16α-Hydroxyprednisolone, an anti-inflammatory drug, could be potentially obtained from hydrocortisone bioconversion by combining a 1,2-dehydrogenation reaction performed by Arthrobacter simplexATCC31652 with a 16α-hydroxylation reaction by Streptomyces roseochromogenes ATCC13400. In this study we tested, for the first time, potential approaches to couple the two reactions using similar pH and temperature conditions for hydrocortisone bioconversion by the two strains. The A. simplex capability to 1,2-dehydrogenate the 16α-hydroxyhydrocortisone, the product of S. roseochromogenes transformation of hydrocortisone, and vice versa the capability of S. roseochromogenes to 16α-hydroxylate the prednisolone were assessed. Bioconversions were studied in shake flasks and strain morphology changes were observed by SEM. Whole cell experiments were set up to perform the two reactions in a sequential mode in alternate order or contemporarily at diverse temperature conditions. A. simplex catalyzed either the dehydrogenation of hydrocortisone into prednisolone efficiently or of 16α-hydroxyhydrocortisone into 16α-hydroxyprednisolone in 24 h (up to 93.9%). Surprisingly S. roseochromogenes partially converted prednisolone back to hydrocortisone. A 68.8% maximum of 16α-hydroxyprednisolone was obtained in 120-h bioconversion by coupling whole cells of the two strains at pH 6.0 and 26 °C. High bioconversion of hydrocortisone into 16α-hydroxyprednisolone was obtained for the first time by coupling A. simplex and S. roseochromogenes.
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Project description:The Streptomyces lividans lsp gene encodes a type II signal peptidase (Lsp) that cleaves the type II leader peptides of lipoproteins. Transcriptional profiling of the bacterium depleted of the lsp gene mainly resulted in deactivation of the sigma U regulon, as well as in downregulation of genes involved in the biogenesis and function of ribosomes and genes encoding some major secretory proteins as determined by hybridisation of commercially available S. lividans genome-wide microarrays. Almost 50% of the dowregulated genes have been described as forming part of the stringent response in streptomycetes. The gene encoding the S. lividans extracellular foldase, the lipoprotein FkpA, is equally downregulated. Therefore, the deletion of lsp from the S. livdans genome temporarily triggers a cellular stress where the stringent response is, at least, partially induced.
2014-05-22 | GSE30728 | GEO
Project description:The gene dagA encoding agarase in Streptomyces coelicolor was cloned in the multicopy plasmid pIJ486 generating plasmid pAGAs5. Plasmid pAGAs5 and pIJ486 were propagated in S. lividans TK21 to obtain S. lividans TK21(pAGAs5) and its isogenic strain S. lividans TK21(pIJ486). Transcriptional profiling of the bacterium that overproduces agarase mainly resulted in the downregulation of the genes involved in the biogenesis and function of ribosomes, together with the downregulation of the genes involved in nitrogen, aminoacids, purine/pyrimidine and central carbon metabolism as well as ABC transporters, redox processes and fatty acids biosynthesis. The number of genes upregulated in the agarase overproducer strain is lower than in the downregulated ones. Almost 50% of the dowregulated genes have been described as forming part of the stringent response in streptomycetes. Therefore, the overproduction of agarase may lead to a condition of nutrient depletion that triggers the stringent response.
2015-07-23 | GSE61447 | GEO
Project description:Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have measured the distribution of the global transcription regulator protein, FNR, across the entire Escherichia coli chromosome in exponentially growing cells. Sixty-three binding targets, each located at the 5' end of a gene, were identified. Some targets are adjacent to poorly transcribed genes where FNR has little impact on transcription. In stationary phase, the distribution of FNR was largely unchanged. Control experiments showed that, like FNR, the distribution of the nucleoid-associated protein, IHF, is little altered when cells enter stationary phase, whilst RNA polymerase undergoes a complete redistribution.
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Project description:DNA sulfur modification is a unique modification occurring in the sugar-phosphate backbone of DNA, with a nonbridging oxygen atom substituted with sulfur in a sequence-specific and Rp stereo-specific manner. Bioinformatics, RNA-seq, and in vitro transcriptional analyses have shown that DNA sulfur modification may be involved in epigenetic regulation. However, the in vivo evidence supporting this assertion is not convincing. Here, we aimed to characterize two sulfur-modified sites near the dndB promoter region in Streptomyces lividans. Single mutation of either site had no effect on dndB transcription, whereas double mutation of both sites significantly elevated dndB expression. These findings suggested that DNA sulfur modification affected gene expression, and the role of DNA sulfur modification in epigenetic regulation depended on the number of sulfur-modified sites. We also identified an inverted repeat, the R repeat sequence, and showed that this sequence participated in the positive regulation of dndB gene expression.
| S-EPMC6186775 | biostudies-literature
Project description:BACKGROUND:Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS:In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION:The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.
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Project description:Various streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Strepotmyces coelicolor A3 (2)] acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli. The expression level in these hosts was two to three times lower than in S. reticuli, indicating the absence of positive regulatory elements. Like S. reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part. The cel-1 gene with its original upstream region was not expressed within Escherichia coli. When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E. coli. However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms. As the cellulase (Avicelase) synthesized by S. reticuli is not cleaved by the E. coli proteases, its posttranslational modification is proposed. With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter.
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Project description:BackgroundAs a polycistronic transcriptional unit of one or more adjacent genes, operons play a key role in regulation and function in prokaryotic biology, and a better understanding of how they are constituted and controlled is needed. Recent efforts have attempted to predict operonic status in sequenced genomes using a variety of techniques and data sources. To date, non-homology based operon prediction strategies have mainly used predicted promoters and terminators present at the extremities of transcriptional unit as predictors, with reasonable success. However, transcription factor binding sites (TFBSs), typically found upstream of the first gene in an operon, have not yet been evaluated.ResultsHere we apply a method originally developed for the prediction of TFBSs in Escherichia coli that minimises the need for prior knowledge and tests its ability to predict operons in E. coli and the 'more complex', pharmaceutically important, Streptomyces coelicolor. We demonstrate that through building genome specific TFBS position-specific-weight-matrices (PSWMs) it is possible to predict operons in E. coli and S. coelicolor with 83% and 93% accuracy respectively, using only TFBS as delimiters of operons. Additionally, the 'palindromicity' of TFBS footprint data of E. coli is characterised.ConclusionTFBS are proposed as novel independent features for use in prokaryotic operon prediction (whether alone or as part of a set of features) given their efficacy as operon predictors in E. coli and S. coelicolor. We also show that TFBS footprint data in E. coli generally contains inverted repeats with significantly (p < 0.05) greater palindromicity than random sequences. Consequently, the palindromicity of putative TFBSs predicted can also enhance operon predictions.
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Project description:The gene aml encoding alpha-amylase in Streptomyces lividans was cloned in the multicopy plasmid pIJ486, generating plasmid pAMI11. Plasmid pAMI11 and pIJ486 were propagated in S. lividans TK21 to obtain S. lividans TK21(pAMI11) and its isogenic strain S. lividans TK21(pIJ486). Transcriptional profiling of the bacterium that overproduces alpha-amylase mainly resulted in the upregulation of genes involved in the biogenesis and function of ribosomes, together with the upregulation of the genes involved in the redox processes, the ABC transporters, the central carbon, aminoacid and purine /pyrimidine metabolism. Moreover, some genes involved in oxidative stress were upregulated. The number of genes downregulated was much lower than the upregulated ones. Therefore, bacteria respond by favouring alpha-amylase overproduction that apparently does not cause damage to the cell.
2015-07-23 | GSE61448 | GEO