Expression of the pstS gene of Streptomyces lividans is regulated by the carbon source and is partially independent of the PhoP regulator

<p>Abstract</p> <p>Background</p> <p>PstS is a phosphate-binding lipoprotein that is part of the high-affinity phosphate transport system. <it>Streptomyces lividans </it>accumulates high amounts of the PstS protein in the supernatant of liquid cultures grown in the presence of different carbon sources, such as fructose or mannose, but not in the presence of glucose or in basal complex medium.</p> <p>Results</p> <p>Functionality experiments revealed that this extracellular PstS protein does not have the capacity to capture phosphate and transfer it to the cell. Regulation of the <it>pstS </it>promoter was studied with Northern blot experiments, and protein levels were detected by Western blot analysis. We observed that the <it>pstS </it>gene was expressed in cultures containing glucose or fructose, but not in complex basal medium. Northern blot analyses revealed that the <it>pst </it>operon (<it>pstSCAB</it>) was transcribed as a whole, although higher transcript levels of <it>pstS </it>relative to those of the other genes of the operon (<it>pstC, pstA </it>and <it>pstB</it>) were observed. Deletion of the -329/-144 fragment of the <it>pstS </it>promoter, including eight degenerated repeats of a sequence of 12 nucleotides, resulted in a two-fold increase in the expression of this promoter, suggesting a regulatory role for this region. Additionally, deletion of the fragment corresponding to the Pho boxes recognized by the PhoP regulator (from nucleotide -141 to -113) resulted in constitutive <it>pstS </it>expression that was independent of this regulator. Thus, the PhoP-independent expression of the <it>pstS </it>gene makes this system different from all those studied previously.</p> <p>Conclusion</p> <p>1.- In <it>S. lividans</it>, only the PstS protein bound to the cell has the capacity to bind phosphate and transfer it there, whereas the PstS form accumulated in the supernatant lacks this capacity. 2.- The stretch of eight degenerated repeats present in the <it>pstS </it>promoter may act as a binding site for a repressor. 3.- There is a basal expression of the <it>pstS </it>gene that is not controlled by the main regulator: PhoP.</p

Identification of the First Functional Toxin-Antitoxin System in Streptomyces

Toxin-antitoxin (TA) systems are widespread among the plasmids and genomes of bacteria and archaea. This work reports the first description of a functional TA system in Streptomyces that is identical in two species routinely used in the laboratory: Streptomyces lividans and S. coelicolor. The described system belongs to the YefM/YoeB family and has a considerable similarity to Escherichia coli YefM/YoeB (about 53% identity and 73% similarity). Lethal effect of the S. lividans putative toxin (YoeBsl) was observed when expressed alone in E. coli SC36 (MG1655 ΔyefM-yoeB). However, no toxicity was obtained when co-expression of the antitoxin and toxin (YefM/YoeBsl) was carried out. The toxic effect was also observed when the yoeBsl was cloned in multicopy in the wild-type S. lividans or in a single copy in a S. lividans mutant, in which this TA system had been deleted. The S. lividans YefM/YoeBsl complex, purified from E. coli, binds with high affinity to its own promoter region but not to other three random selected promoters from Streptomyces. In vivo experiments demonstrated that the expression of yoeBsl in E. coli blocks translation initiation processing mRNA at three bases downstream of the initiation codon after 2 minutes of induction. These results indicate that the mechanism of action is identical to that of YoeB from E. coli

Identification of the First Functional Toxin-Antitoxin System in Streptomyces

Toxin-antitoxin (TA) systems are widespread among the plasmids and genomes of bacteria and archaea. This work reports the first description of a functional TA system in Streptomyces that is identical in two species routinely used in the laboratory: Streptomyces lividans and S. coelicolor. The described system belongs to the YefM/YoeB family and has a considerable similarity to Escherichia coli YefM/YoeB (about 53% identity and 73% similarity). Lethal effect of the S. lividans putative toxin (YoeBsl) was observed when expressed alone in E. coli SC36 (MG1655 ΔyefM-yoeB). However, no toxicity was obtained when co-expression of the antitoxin and toxin (YefM/YoeBsl) was carried out. The toxic effect was also observed when the yoeBsl was cloned in multicopy in the wild-type S. lividans or in a single copy in a S. lividans mutant, in which this TA system had been deleted. The S. lividans YefM/YoeBsl complex, purified from E. coli, binds with high affinity to its own promoter region but not to other three random selected promoters from Streptomyces. In vivo experiments demonstrated that the expression of yoeBsl in E. coli blocks translation initiation processing mRNA at three bases downstream of the initiation codon after 2 minutes of induction. These results indicate that the mechanism of action is identical to that of YoeB from E. coli

Two-component systems in Streptomyces: key regulators of antibiotic complex pathways

Streptomyces, the main antibiotic-producing bacteria, responds to changing environmental conditions through a complex sensing mechanism and two-component systems (TCSs) play a crucial role in this extraordinary “sensing” device. Moreover, TCSs are involved in the biosynthetic control of a wide range of secondary metabolites, among them commercial antibiotics. Increased knowledge about TCSs can be a powerful asset in the manipulation of bacteria through genetic engineering with a view to obtaining higher efficiencies in secondary metabolite production. In this review we summarise the available information about Streptomyces TCSs, focusing specifically on their connections to antibiotic production

Characterization of Actinomycetes Strains Isolated from the Intestinal Tract and Feces of the Larvae of the Longhorn Beetle Cerambyx welensii

[EN]Actinomycetes constitute a large group of Gram-positive bacteria present in different habitats. One of these habitats involves the association of these bacteria with insects. In this work, we have studied twenty-four actinomycetes strains isolated from the intestinal tract and feces from larvae of the xylophagous coleopteran Cerambyx welensii and have shown that seventeen strains present hydrolytic activity of some of the following substrates: cellulose, hemicellulose, starch and proteins. Fourteen of the isolates produce antimicrobial molecules against the Gram-positive bacteria Micrococcus luteus. Analysis of seven strains led us to identify the production of a wide number of compounds including streptanoate, alpiniamide A, alteramides A and B, coproporphyrin III, deferoxamine, demethylenenocardamine, dihydropicromycin, nocardamine, picromycin, surugamides A, B, C, D and E, tirandamycins A and B, and valinomycin. A significant number of other compounds, whose molecular formulae are not included in the Dictionary of Natural Products (DNP), were also present in the extracts analyzed, which opens up the possibility of identifying new active antibiotics. Molecular identification of ten of the isolated bacteria determined that six of them belong to the genus Streptomyces, two of them are included in the genus Amycolatopsis and two in the genus Nocardiopsis

Divulgación en tiempos de pandemia: el poder de las webs

Trabajo presentado en el simposio Micromundo Aprendizaje-Servicio para el descubrimiento de antibiótico

An Experimental DUAL Model of Advanced Liver Damage

Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcohol-associated and metabolic-associated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcohol-associated liver disease plus metabolic-associated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNA-sequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets.Supported by EXOHEP-CM (S2017/BMD-3727), Ramón y Cajal (RYC-2014-15242 and RYC-2015-17438), NanoLiver-CM (Y2018/NMT-4949), COST Action (CA17112), AMMF (2018/117), ERAB (EA 18/14), MINECO Retos (SAF2016-78711 and SAF2017-87919-R), and German Research Foundation (DFG NE 2128/2-1, SFB 1382-403224013/A02, and SFB/TRR57/P04). FJC is a Gilead Research Liver Scholar. The research group belongs to the validated Research group Ref. 970935 “Liver Pathophysiology”, 920631 “Lymphocyte immunology”, 920361 “Immunogenética e inmunología de las mucosas” and IBL-6 (imas12-associated). FG and KZ are Chinese Scholarship Council (CSC) fellows. O.E.-V is supported by Beca FPI (associated to MINECO SAF2017-87919R) and R.B.-U. by Contratos predoctorales de personal investigador en formación UCM-Banco Santander (CT63/19)

Universidad y sociedad: comunicación e integración en empresas e instituciones públicas y organizaciones no lucrativas. Nuevos avances

Depto. de Teorías y Análisis de la ComunicaciónFac. de Ciencias de la InformaciónFALSEsubmitte

Novel Two-Component Systems Implied in Antibiotic Production in Streptomyces coelicolor

The abundance of two-component systems (TCSs) in Streptomyces coelicolor A3(2) genome indicates their importance in the physiology of this soil bacteria. Currently, several TCSs have been related to antibiotic regulation, and the purpose in this study was the characterization of five TCSs, selected by sequence homology with the well-known absA1A2 system, that could also be associated with this important process. Null mutants of the five TCSs were obtained and two mutants (ΔSCO1744/1745 and ΔSCO4596/4597/4598) showed significant differences in both antibiotic production and morphological differentiation, and have been renamed as abr (antibiotic regulator). No detectable changes in antibiotic production were found in the mutants in the systems that include the ORFs SCO3638/3639, SCO3640/3641 and SCO2165/2166 in any of the culture conditions assayed. The system SCO1744/1745 (AbrA1/A2) was involved in negative regulation of antibiotic production, and acted also as a negative regulator of the morphological differentiation. By contrast, the system SCO4596/4597/4598 (AbrC1/C2/C3), composed of two histidine kinases and one response regulator, had positive effects on both morphological development and antibiotic production. Microarray analyses of the ΔabrC1/C2/C3 and wild-type transcriptomes revealed downregulation of actII-ORF4 and cdaR genes, the actinorhodin and calcium-dependent antibiotic pathway-specific regulators respectively. These results demonstrated the involvement of these new two-component systems in antibiotic production and morphological differentiation by different approaches. One is a pleiotropic negative regulator: abrA1/A2. The other one is a positive regulator composed of three elements, two histidine kinases and one response regulator: abrC1/C2/C3