Laura Sevillano

Proteome Changes in Tomato Fruits Prior to Visible Symptoms of Chilling Injury are Linked to Defensive Mechanisms, Uncoupling of Photosynthetic Processes and Protein Degradation Machinery

A comparative proteomic analysis between tomato fruits stored at chilling and non-chilling temperatures was carried out just before the appearance of visible symptoms of chilling injury. At this stage of the stress period it was possible to discriminate between proteins involved in symptoms and proteins implicated in response. To investigate the changes in the tomato fruit proteome under this specific stressful condition, two-dimensional differential in-gel electrophoresis coupled with spot identification by mass spectrometry was applied. This proteomic approach allowed the identification of differentially expressed proteins which are involved in two main biological functions: (i) defensive mechanisms represented by small heat shock and late embryogenesis proteins; and (ii) reaction to the uncoupling of photosynthetic processes and the protein degradation machinery. One of the first changes observed in chilled fruits is the down-regulation of ATP synthase, 26S proteasome subunit RPN11 and aspartic proteinase, whereas the first responses in order to deal with the stress are mainly multifunctional proteins involved not only in metabolism but also in stress regulation such as glyceraldehyde phosphate dehydrogenase, 2-oxoglutarate dehydrogenase and invertase. In addition, our data seem to indicate a possible candidate to be used as a protein marker for further studies on cold stress: aldose-1-epimerase, which seems to have an important role in low temperature tolerance.

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.

Stable expression plasmids for Streptomyces based on a toxin-antitoxin system

BackgroundBacteria included in the genus Streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. One of them is that some of its species have a high secretion capacity and hence the protein of interest could be released to the culture supernatant, facilitating downstream processing. To date, all the expression vectors described for these bacteria contain antibiotic resistance genes as selection markers. However, the use of antibiotics to produce proteins at industrial level is currently becoming more restricted owing to the possibility of contamination of the final product. In this report, we describe the use of the S. lividans yefM/yoeBsl toxin-antitoxin system to develop a stable plasmid expression system.ResultsIn order to use the yefM/yoeBsl system to stabilize expression plasmids in Streptomyces, a S. lividans mutant strain that contained only the toxin gene (yoeBsl) in its genome and the antitoxin gene (yefMsl) located in a temperature-sensitive plasmid was constructed and used as host. This strain was transformed with an expression plasmid harbouring both the antitoxin gene and the gene encoding the protein of interest. Thus, after elimination of the temperature-sensitive plasmid, only cells with the expression plasmid were able to survive. On using this system, two proteins - an α-amylase from S. griseus and a xylanase from S. halstedii - were overproduced without the addition of antibiotic to the culture medium. The production of both proteins was high, even after long incubations (8 days), and after serial subcultures, confirming the stability of the plasmids without antibiotic selection.ConclusionsThis is the first report that describes the use of a toxin-antitoxin system to maintain high -copy plasmids in Streptomyces. This finding could be a valuable tool for using Streptomyces as a host to produce proteins at the industrial and pharmaceutical levels without the use of antibiotics in the production step.

The contribution of biotechnology to improving post-harvest chilling tolerance in fruits and vegetables using heat-shock proteins

Fresh fruits and vegetables have a short post-harvest life and are prone to post-harvest losses due to mechanical injury, physiological causes and decay. Low-temperature storage is widely used as post-harvest treatment applied for delaying senescence in vegetables and ornamentals and ripening in fruits, upholding their post-harvest quality. But the refrigerated storage of tropical and subtropical crop plant species provokes a set of physiological alterations known as chilling injury that negatively affect their quality and frequently renders the product not saleable. Membrane damage and reactive oxygen species (ROS) accumulation are the main adverse effects of chilling injury impact in sensitive horticultural products. The chilling injury tolerance of certain plant species is attributed to their ability to accumulate heat-shock proteins (HSP). The beneficial action of HSP in chilling tolerance is due to their chaperone activity but, besides this biological function, small HSP (sHSP) are able to function as membrane stabilizers and ROS scavengers, or synergistically with cell antioxidant systems. Also, biosynthesis of osmolytes such as raffinose and proline is under the regulation of heat-shock transcription factors (HSTF). These molecules are critical for osmotic adjustment since low temperatures also provoke a secondary osmotic stress. The use of biotechnological strategies can be envisaged, with the aim of generating engineered crop plants of horticultural interest to induce the production and action of HSP and HSTF, in order to assure the beneficial effects of these proteins in promoting chilling injury tolerance during their post-harvest refrigerated storage. In particular, induction of HSTF expression using biotechnology has significant potential and interest for reducing the impact of chilling injury on sensitive produce, avoiding the practical difficulties of applying the classic post-harvest technologies based on heat treatment.

The Orphan Response Regulator Aor1 Is a New Relevant Piece in the Complex Puzzle of Streptomyces coelicolor Antibiotic Regulatory Network

Streptomyces coelicolor, the best-known biological antibiotic producer, encodes 29 predicted orphan response regulators (RR) with a putative role in the response to environmental stimuli. However, their implication in relation to secondary metabolite production is mostly unexplored. Here, we show how the deletion of the orphan RR Aor1 (SCO2281) provoked a drastic decrease in the production of the three main antibiotics produced by S. coelicolor and a delay in morphological differentiation. With the aim to better understand the transcriptional events underpinning these phenotypes, and the global role of Aor1 in Streptomyces, a transcriptional fingerprint of the Δaor1 mutant was compared to a wild-type strain. RNA-Seq analysis revealed that the deletion of this orphan regulator affects a strikingly high number of genes, such as the genes involved in secondary metabolism, which matches the antibiotic production profiles observed. Of particular note, the sigma factor SigB and all of the genes comprising its regulon were up regulated in the mutant. Our results show that this event links osmotic stress to secondary metabolite production in S. coelicolor and indicates that the RR encoded by aor1 could be a key regulator in both of these processes.

Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces

BackgroundThe industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential.ResultsIn this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed.ConclusionsThe total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described.Graphical abstractAntibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.

Empleo de Rocas Ornamentales en Época Romana en la Región de Murcia

This work was supported by the projects: CICYT (AGL 2008- 05258-CO2-1-2- AGR and AGL 2011-30022-CO2-01-02), Fundacion Seneca (15356/PI/10) and Vias y Construccion, S. A. (CDTI IDI-20101191).Resumen del trabajo presentado a la 4th International Conference on Analytical proteomics, celebrada en Caparica (Portugal) del 7 al 9 de septiembre de 2015.Resumen del trabajo presentado al XXII Congreso Espanol de Toxicologia y VI Iberoamericano, celebrado en Valencia (Espana) del 28 al 30 de junio de 2017.