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Streptomyces coelicolor

José L Neira, Felipe Hornos, Concetta Cozza, Ana Cámara-Artigas, Olga Abián, Adrián Velázquez-Campoy
The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ70 factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPrsc, and E...
December 26, 2017: Archives of Biochemistry and Biophysics
Ryan Cook, Drew Hannon, Jonathan N Southard, Sudipta Majumdar
A one semester undergraduate biochemistry laboratory experience is described for an understanding of recombinant technology from gene cloning to protein characterization. An integrated experimental design includes three sequential modules: molecular cloning, protein expression and purification, and protein analysis and characterization. Students perform the tasks of cloning, expression, purification, analysis, and characterization of small laccase (SLAC) from Streptomyces coelicolor. SLAC is an extremely robust well-characterized protein/enzyme, which serves as an ideal model for undergraduate teaching laboratories...
December 23, 2017: Biochemistry and Molecular Biology Education
Rui Zhang, Dana M Lord, Rakhi Bajaj, Wolfgang Peti, Rebecca Page, Jason K Sello
In Streptomyces coelicolor, we identified a para-hydroxybenzoate (PHB) hydroxylase, encoded by gene pobA (SCO3084), which is responsible for conversion of PHB into PCA (protocatechuic acid), a substrate of the β-ketoadipate pathway which yields intermediates of the Krebs cycle. We also found that the transcription of pobA is induced by PHB and is negatively regulated by the product of SCO3209, which we named PobR. The product of this gene is highly unusual in that it is the apparent fusion of two IclR family transcription factors...
December 12, 2017: Nucleic Acids Research
Giuseppe Gallo, Andrea Scaloni
Proteomics based on 2D-Difference In Gel Electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) procedures can be considered a "gold standard" to determine quantitatively and comparatively protein abundances in cell extracts from different biological sources/conditions according to a gel-based approach. In particular, 2D-DIGE is used for protein specie separation, detection, and relative quantification, whenever tandem MS is used to obtain peptide sequence information that is managed according to bioinformatic procedures to identify the differentially represented protein species...
2018: Methods in Molecular Biology
Deepti Yadav, Bibhuti Ranjan, Nokuthula Mchunu, Marilize Le Roes-Hill, Tukayi Kudanga
This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500±10U/l), which were further increased seven fold by pre-incubation at 80°C for 30min. The enzyme (∼38kDa) had an optimum activity at 80°C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142...
December 1, 2017: International Journal of Biological Macromolecules
Bernhard Kepplinger, Stephanie Morton-Laing, Kenneth Holst Seistrup, Emma Claire Louise Marrs, Adam Paul Hopkins, John David Perry, Henrik Strahl, Michael John Hall, Jeff Errington, Nicholas Edward Ellis Allenby
Antibiotics that interfere with the bacterial cytoplasmic membrane have long term potential for the treatment of infectious diseases, as this mode of action is anticipated to result in low resistance frequency. Vancoresmycin is an understudied natural product antibiotic consisting of a terminal tetramic acid moiety fused to a linear, highly oxygenated, stereochemically complex polyketide chain. Vancoresmycin shows minimum inhibitory concentrations (MICs) from 0.125 to 2 µg/mL against a range of clinically relevant, antibiotic-resistant Gram-positive bacteria...
November 29, 2017: ACS Chemical Biology
Yaojun Tong, Helene Lunde Robertsen, Kai Blin, Tilmann Weber, Sang Yup Lee
Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editing methods, some strains even cannot be manipulated at all. This restricts systematic metabolic engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing...
2018: Methods in Molecular Biology
Alvaro R Lara, Karim E Jaén, Juan-Carlos Sigala, Lars Regestein, Jochen Büchs
Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTRmax) of 7 and 11 mmol L(-1) h(-1)...
2017: Journal of Biological Engineering
Jessy Mariam, Ruchi Anand
Fluorescence spectroscopy is an important analytical tool which is widely employed to study biological systems. This technique can be applied to qualitatively and quantitatively probe protein-ligand interactions primarily because of its sensitivity, selectivity, nondestructive and rapid form of analysis. In this chapter we describe the utility of this technique to establish a label-free, universal screening protocol for putative γ-butyrolactone (GBL) receptors by exploiting the intrinsic fluorescence of a highly conserved tryptophan residue that constitutes the hydrophobic pocket for GBL binding, a unique feature possessed by this family of receptors...
2018: Methods in Molecular Biology
Marc Biarnes-Carrera, Rainer Breitling, Eriko Takano
In Streptomyces, the onset of antibiotic production and sporulation is coordinated through small diffusible molecules known as γ-butyrolactones (GBLs). These are active in very low amounts, and their extraction and characterization are challenging. Here we describe a rapid, small-scale method for the extraction of GBL from Streptomyces coelicolor, from both solid and liquid cultures, which provides sufficient material for subsequent bioassays and partial characterization. We also present two different bioassay techniques for the detection and quantification of the GBL content in the extracts: the antibiotic bioassay and the kanamycin bioassay...
2018: Methods in Molecular Biology
Feng Xu, Jin Wang, Guo-Ping Zhao
It has been known that some Streptomyces species, including the model strain Streptomyces coelicolor, are vulnerable to visible light. Much evidence demonstrated that the phototoxicity induced by visible light is a consequence of the formation of intracellular reactive oxygen species (ROS), which are potentially harmful to cells. In this study, we found that α-ketoglutarate (α-KG) has a protective role against the phototoxicity in S. coelicolor. It could be because that α-KG can detoxify the ROS with the concomitant formation of succinate, which mediates the cells getting into anaerobiosis to produce more NADH and maintain intracellular redox homeostasis, a situation that was demonstrated by overexpressing gdhA in S...
March 2017: Biochemistry and Biophysics Reports
Seomara Martín-Martín, Antonio Rodríguez-García, Fernando Santos-Beneit, Etelvina Franco-Domínguez, Alberto Sola-Landa, Juan Francisco Martín
Phosphate control of the biosynthesis of secondary metabolites in Streptomyces is mediated by the two component system PhoR-PhoP. Linked to the phoR-phoP cluster, and expressed in the opposite orientation, is a phoU-like encoding gene with low identity to the phoU gene of Escherichia coli. Expression of this phoU-like gene is strictly dependent on PhoP activation. We have isolated a PhoU-null mutant and used transcriptomic and RNA-sequencing (RNA-seq) procedures to identify its transcription start site and regulation...
November 1, 2017: Journal of Antibiotics
Jae Woong Choi, Sung Sun Yim, Ki Jun Jeong
In the cell surface display, the choice of host cell and anchoring motif are the most crucial for the efficient display of passenger proteins. Corynebacterium glutamicum has mycolic acid layer in outer membrane and the use of protein in the mycolic acid layer as an anchoring motif can provide a potential platform for surface display in C. glutamicum. All 19 mycolic acid layer proteins of C. glutamicum are analyzed, and two proteins, NCgl0535 and NCgl1337, which have a signal peptide and predicted O-mycoloylation site, are selected as anchoring motifs candidates...
October 26, 2017: Biotechnology Journal
Jiafang Fu, Gongli Zong, Peipei Zhang, Junxia Ma, Xiuhua Pang, Guangxiang Cao
The xdhR gene encodes a TetR-family regulator in Streptomyces coelicolor. However, little is known about the function of XdhR in regulating actinorhodin production. Here, we report that XdhR negatively regulates actinorhodin biosynthesis in S. coelicolor. Deletion of xdhR resulted in overproduction of actinorhodin by approximately 2.5-fold compared to the wild-type strain. Complementation of the xdhR deletion strain restored actinorhodin production to normal levels. In addition, the relative expression levels of actinorhodin cluster genes were all significantly increased in the xdhR deletion strain compared to the wild-type strain...
October 24, 2017: FEMS Microbiology Letters
Ying Xu, Di You, Bang-Ce Ye
N-Lysine acetylation is a dynamic, reversible and regulatory post-translational modification (PTM) in prokaryotes, which integrates and coordinates metabolisms responding to environmental clues. However, the molecular mechanism underlying the signalling pathway from nutrient sensing to protein acetylation remains incompletely understood in micro-organisms. Here we found that global nitrogen regulator GlnR directly controls transcription of genes encoding lysine deacetylases in Actinobacteria. Electrophoretic mobility shift assays and real-time PCR (RT-PCR) in three Actinobacteria species (Saccharopolyspora erythraea, Streptomyces coelicolor and Mycobacterium smegmatis) revealed that GlnR regulator protein is able to interact with the promoter regions of these genes and activate their transcription...
October 23, 2017: Microbiology
Hu Wang, Guoping Zhao, Xiaoming Ding
Growth of Streptomyces in submerged culture is characterized by the formation of complex mycelial particles, known as pellets or clumps, which strongly influence antibiotic production. Also, many bioactive molecules produced by Streptomyces have great potential to modulate soil bacteria morphological development. However, there has been no effort directed at engineering mycelial morphology using these small molecules. Here, thiostrepton was identified, using a combination of qRT-PCR, semi-preparative HPLC, and MALDI-TOF MS, as a pellet-inducing compound produced by S...
October 16, 2017: Scientific Reports
Mina Dokouhaki, Andrew Hung, Emma L Prime, Greg G Qiao, Li Day, Sally L Gras
Chaplin E, or Chp E, is a surface active peptide secreted by Streptomyces coelicolor that adopts different structures depending on solution pH but the effect of these structures on the interfacial properties of Chp E is not known. In experiments paired with simulations, Chp E was found to display pH-dependent interfacial assembly and surface activity. At pH 3.0, Chp E formed an ordered non-amyloidal interfacial film with high surface activity; while at pH 10.0, Chp E self-assembled into a heterogeneous film containing randomly arranged fibrils at the interface that was less surface active compared to the film formed at pH 3...
October 4, 2017: Colloids and Surfaces. B, Biointerfaces
Jack E Bowyer, Emmanuel Lc de Los Santos, Kathryn M Styles, Alex Fullwood, Christophe Corre, Declan G Bates
BACKGROUND: The antibiotic methylenomycin A is produced naturally by Streptomyces coelicolor A3(2), a model organism for streptomycetes. This compound is of particular interest to synthetic biologists because all of the associated biosynthetic, regulatory and resistance genes are located on a single cluster on the SCP1 plasmid, making the entire module easily transferable between different bacterial strains. Understanding further the regulation and biosynthesis of the methylenomycin producing gene cluster could assist in the identification of motifs that can be exploited in synthetic regulatory systems for the rational engineering of novel natural products and antibiotics...
2017: Journal of Biological Engineering
María Ordóñez-Robles, Fernando Santos-Beneit, Silvia M Albillos, Paloma Liras, Juan F Martín, Antonio Rodríguez-García
In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces tsukubaensis. A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster...
October 5, 2017: Applied Microbiology and Biotechnology
Agnieszka Strzalka, Marcin J Szafran, Terence Strick, Dagmara Jakimowicz
Streptomyces topoisomerase I (TopA) exhibits exceptionally high processivity. The enzyme, as other actinobacterial topoisomerases I, differs from its bacterial homologs in its C-terminal domain (CTD). Here, bioinformatics analyses established that the presence of lysine repeats is a characteristic feature of actinobacterial TopA CTDs. Streptomyces TopA contains the longest stretch of lysine repeats, which terminate with acidic amino acids. DNA-binding studies revealed that the lysine repeats stabilized the TopA-DNA complex, while single-molecule experiments showed that their elimination impaired enzyme processivity...
November 16, 2017: Nucleic Acids Research
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