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BACKGROUND: Several two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor. RESULTS: The ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869. CONCLUSION: This bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces.

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The co-culturing of microorganisms is a well-known strategy to study microbial interactions in the laboratory. This approach facilitates the identification of new signals and molecules produced by one species that affects other species' behavior. In this work, we have studied the effects of the interaction of nine Streptomyces species (S. albidoflavus, S. ambofaciens, S. argillaceus, S. griseus, S. lividans, S. olivaceus, S. parvulus, S. peucetius, and S. rochei) with the predator bacteria Myxococcus xanthus, five of which (S. albidoflavus, S. griseus, S. lividans, S. olivaceus, and S. argillaceus) induce mound formation of M. xanthus on complex media (Casitone Yeast extract (CYE) and Casitone tris (CTT); media on which M. xanthus does not form these aggregates under normal culture conditions. An in-depth study on S. griseus-M. xanthus interactions (the Streptomyces strain producing the strongest effect) has allowed the identification of two siderophores produced by S. griseus, demethylenenocardamine and nocardamine, responsible for this grouping effect over M. xanthus. Experiments using pure commercial nocardamine and different concentrations of FeSO4 show that iron depletion is responsible for the behavior of M. xanthus. Additionally, it was found that molecules, smaller than 3 kDa, produced by S. peucetius can induce the production of DK-xanthenes by M. xanthus.

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Bacteria of the genus Streptomyces have a plethora of transcriptional regulators, among which the xenobiotic response element (XRE) plays an important role. In this organism, XRE regulators are often followed downstream by small proteins of unknown function containing a DUF397 domain. It has been proposed that XRE/DUF397 pairs constitute type II toxin-antitoxin (TA) systems. However, previous work carried out by our group has shown that one of these systems is a strong activator of antibiotic production in S. coelicolor and other Streptomyces species. In this work, we have studied the overexpression of fourteen XRE/DUF397 pairs present in the S. coelicolor genome and found that none behave as a type II TA system. Instead, they act as pleiotropic regulators affecting, in a dependent manner, antibiotic production and morphological differentiation on different culture media. After deleting, individually, six XRE/DUF397 pairs (those systems producing more notable phenotypic changes when overexpressed: SCO2246/45, SCO2253/52, SCO4176/77, SCO4678/79, SCO6236/35, and SCO7615/16), the pair SCO7615/16 was identified as producing the most dramatic differences as compared to the wild-type strain. The SCO7615/16 mutant had a different phenotype on each of the media tested (R2YE, LB, NMMP, YEPD, and MSA). In particular, on R2YE and YEPD media, a bald phenotype was observed even after 7 days, with little or no actinorhodin (ACT) production. Lower ACT production was also observed on LB medium, but the bacteria were able to produce aerial mycelium. On NMMP medium, the mutant produced a larger amount of ACT as compared with the wild-type strain.

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Bacteria of the Streptomyces genus constitute an authentic biotech gold mine thanks to their ability to produce a myriad of compounds and enzymes of great interest at various clinical, agricultural, and industrial levels. Understanding the physiology of these organisms and revealing their regulatory mechanisms is essential for their manipulation and application. Two-component systems (TCSs) constitute the predominant signal transduction mechanism in prokaryotes, and can detect a multitude of external and internal stimuli and trigger the appropriate cellular responses for adapting to diverse environmental conditions. These global regulatory systems usually coordinate various biological processes for the maintenance of homeostasis and proper cell function. Here, we review the multiple TCSs described and characterized in Streptomyces coelicolor, one of the most studied and important model species within this bacterial group. TCSs are involved in all cellular processes; hence, unravelling the complex regulatory network they form is essential for their potential biotechnological application.

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Streptomyces bacteria produce a wide number of antibiotics and antitumor compounds that have attracted the attention of pharmaceutical and biotech companies. In this study, we provide evidence showing that the xylem sap from grapevines has a positive effect on the production of different antibiotics by several Streptomyces species, including S. ambofaciens ATCC 23877 and S. argillaceus ATCC 12596 among others. The production of several already known compounds was induced: actinomycin D, chromomycin A3, fungichromin B, mithramycin A, etc., and four compounds with molecular formulas not included in the Dictionary of Natural Products (DNP v28.2) were also produced. The molecules present in the xylem sap that acts as elicitors were smaller than 3 kDa and soluble in water and insoluble in ether, ethyl acetate, or methanol. A combination of potassium citrate and di-D-fructose dianhydrides (related to levanbiose or inulobiose) seemed to be the main effectors identified from the active fraction. However, the level of induction obtained in the presence of these compounds mix was weaker and delayed with respect to the one got when using the whole xylem sap or the 3 kDa sap fraction, suggesting that another, not identified, elicitor must be also implied in this induction.

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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.

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Antibiotic resistance currently presents one of the biggest threats to humans. The development and implementation of strategies against the spread of superbugs is a priority for public health. In addition to raising social awareness, approaches such as the discovery of new antibiotic molecules and the elucidation of resistance mechanisms are common measures. Accordingly, the two-component system (TCS) of Streptomyces coelicolor AbrB1/B2, offer amenable ways to study both antibiotic production and resistance. Global transcriptomic comparisons between the wild-type strain S. coelicolor M145 and the mutant ΔabrB, using RNA-Seq, showed that the AbrB1/B2 TCS is implicated in the regulation of different biological processes associated with stress responses, primary and secondary metabolism, and development and differentiation. The ΔabrB mutant showed the up-regulation of antibiotic biosynthetic gene clusters and the down-regulation of the vancomycin resistance gene cluster, according to the phenotypic observations of increased antibiotic production of actinorhodin and undecylprodigiosin, and greater susceptibility to vancomycin. The role of AbrB1/B2 in vancomycin resistance has also been shown by an in silico analysis, which strongly indicates that AbrB1/B2 is a homolog of VraR/S from Staphylococcus aureus and LiaR/S from Enterococcus faecium/Enterococcus faecalis, both of which are implied in vancomycin resistance in these pathogenic organisms that present a serious threat to public health. The results obtained are interesting from a biotechnological perspective since, on one hand, this TCS is a negative regulator of antibiotic production and its high degree of conservation throughout Streptomyces spp. makes it a valuable tool for improving antibiotic production and the discovery of cryptic metabolites with antibiotic action. On the other hand, AbrB1/B2 contributes to vancomycin resistance and is a homolog of VraR/S and LiaR/S, important regulators in clinically relevant antibiotic-resistant bacteria. Therefore, the study of AbrB1/B2 could provide new insight into the mechanism of this type of resistance.

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The xenobiotic response element (XRE) transcription factors belong to a regulator family frequently found in Streptomyces that are often followed by small proteins with a DUF397 domain. In fact, the pair XRE-DUF397 has been proposed to comprise toxin-antitoxin (TA) type II systems. In this work, we demonstrate that one of these putative TA-systems, encoded by the genes SCO4441 and SCO4442 of Streptomyces coelicolor, and denominated Scr1/Scr2 (which stands for S. c oelicolor r egulator), does not behave as a toxin-antitoxin system under the conditions used as was originally expected. Instead the pair Scr1/Scr2 acts as a strong positive regulator of endogenous antibiotic production in S. coelicolor. The analysis of the 19 Streptomyces strains tested determined that overexpression of the pair Scr1/Scr2 drastically induces the production of antibiotics not only in S. coelicolor, but also in Streptomyces lividans, Streptomyces peucetius, Streptomyces steffisburgensis and Streptomyces sp. CA-240608. Our work also shows that Scr1 needs Scr2 to exert positive regulation on antibiotic production.

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Sequencing of Streptomyces genomes has revealed they harbor a high number of biosynthesis gene cluster (BGC), which uncovered their enormous potentiality to encode specialized metabolites. However, these metabolites are not usually produced under standard laboratory conditions. In this manuscript we report the activation of BGCs for antimycins, carotenoids, germicidins and desferrioxamine compounds in Streptomyces argillaceus, and the identification of the encoded compounds. This was achieved by following different strategies, including changing the growth conditions, heterologous expression of the cluster and inactivating the adpAa or overexpressing the abrC3 global regulatory genes. In addition, three new carotenoid compounds have been identified.

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Xylooligosaccharides display interesting prebiotic effects on human health. The endoxylanase Xys1Δ, from Streptomyces halstedii JM8, was immobilized and stabilized on glyoxyl-agarose beads by multipoint covalent attachment using a novel strategy based on surface coating with a multilayer of polymers. The optimal modification consisted of surface coating with a bilayer formed by a layer of derived dextran polymers and a layer of polyethylenimine. The optimized biocatalyst was 550-fold more stable than one-point covalent immobilized Xys1Δ (at 70 °C, pH 7). This biocatalyst was tested for the production of xylooligosaccharides from soluble xylans from various sources. Hydrolysis of beechwood, wheat straw and corncob xylans was 93% in 4 h, 44% in 5 h and 100% in 1 h, respectively. Maximum values of xylooligosaccharides were found for beechwood at 20.6 mg/mL, wheat at 12.5 mg/mL and corncob at 30.4 mg/mL. The optimized biocatalyst was reused for 15 reaction cycles without affecting its catalytic activity.

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BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: The 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 abstract Antibiotic 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.

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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.

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BACKGROUND: Actinomycetes are saprophytic soil bacteria, and a rich source of industrial enzymes. While some of these enzymes can be produced using well-characterized production platforms such as Escherichia coli or Bacillus subtilis, Streptomyces lividans may be the preferred host for proper folding and efficient secretion of active enzymes. A combination of promoters, signal peptides and hosts were tested in order to obtain the best protein expression in this actinomycete. The xylanase, Xys1, from S. halstedii, the α-amylase, Amy, from S. griseus and the small laccase, SLAC, from S. coelicolor were used as reporters. RESULTS: The promoters xysAp from S. halstedii JM8 and pstSp from S. lividans were the most efficient among those tested. An improvement of 17 % was obtained in xylanase activity when the signal peptide of the α-amylase protein (Amy) of S. griseus IMRU3570 was used to direct its secretion. Enhanced expression of SsgA, a protein that plays a role in processes that require cell-wall remodelling, resulted in a improvement of 40 and 70 % of xylanase and amylase production, respectively. Deletion of genes SLI7232 and SLI4452 encoding putative repressors of xysAp provided improvement of production up to 70 % in the SLI7232 deletion strain. However, full derepression of this promoter activity was not obtained under the conditions assayed. CONCLUSIONS: Streptomyces lividans is a frequently used platform for industrial enzyme production and a rational strain-development approach delivered significant improvement of protein production by this host.

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Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. qRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites.

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Emergence of antibiotic resistant pathogens is changing the way scientists look for new antibiotic compounds. This race against the increased prevalence of multi-resistant strains makes it necessary to expedite the search for new compounds with antibiotic activity and to increase the production of the known. Here, we review a variety of new scientific approaches aiming to enhance antibiotic production in Streptomyces. These include: (i) elucidation of the signals that trigger the antibiotic biosynthetic pathways to improve culture media, (ii) bacterial hormone studies aiming to reproduce intra and interspecific communications resulting in antibiotic burst, (iii) co-cultures to mimic competition-collaboration scenarios in nature, and (iv) the very recent in situ search for antibiotics that might be applied in Streptomyces natural habitats. These new research strategies combined with new analytical and molecular techniques should accelerate the discovery process when the urgency for new compounds is higher than ever.

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The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant ΔabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the ΔabrA1/A2 strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.

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The atypical two-component system (TCS) AbrC1/C2/C3 (encoded by SCO4598, SCO4597, and SCO4596), comprising two histidine kinases (HKs) and a response regulator (RR), is crucial for antibiotic production in Streptomyces coelicolor and for morphological differentiation under certain nutritional conditions. In this study, we demonstrate that deletion of the RR-encoding gene, abrC3 (SCO4596), results in a dramatic decrease in actinorhodin (ACT) and undecylprodiginine (RED) production and delays morphological development. In contrast, the overexpression of abrC3 in the parent strain leads to a 33% increase in ACT production in liquid medium. Transcriptomic analysis and chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis of the ΔabrC3 mutant and the parent strain revealed that AbrC3 directly controls ACT production by binding to the actII-ORF4 promoter region; this was independently verified by in vitro DNA-binding assays. This binding is dependent on the sequence 5'-GAASGSGRMS-3'. In contrast, the regulation of RED production is not due to direct binding of AbrC3 to either the redZ or redD promoter region. This study also revealed other members of the AbrC3 regulon: AbrC3 is a positive autoregulator which also binds to the promoter regions of SCO0736, bdtA (SCO3328), absR1 (SCO6992), and SCO6809. The direct targets share the 10-base consensus binding sequence and may be responsible for some of the phenotypes of the ΔabrC3 mutant. The identification of the AbrC3 regulon as part of the complex regulatory network governing antibiotic production widens our knowledge regarding TCS involvement in control of antibiotic synthesis and may contribute to the rational design of new hyperproducer host strains through genetic manipulation of such systems.

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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.

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BACKGROUND: Bacteria 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. RESULTS: In 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. CONCLUSIONS: This 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.

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Phosphate metabolism regulates most of the life processes of microorganisms. In the present work we obtained and studied a Streptomyces lividans ppk/pstS double mutant, which lacks polyphosphate kinase (PPK) and the high-affinity phosphate-binding protein (PstS), impairing at the same time the intracellular storage of polyphosphate and the intake of new inorganic phosphate from a phosphate-limited medium, respectively. In some of the aspects analyzed, the ppk/pstS double mutant was more similar to the wt strain than was the single pstS mutant. The double mutant was thus able to grow in phosphate-limited media, whereas the pstS mutant required the addition of 1 mM phosphate under the assay conditions used. The double mutant was able to incorporate more than one fourth of the inorganic phosphate incorporated by the wt strain, whereas phosphate incorporation was almost completely impaired in the pstS mutant. Noteworthy, under phosphate limitation conditions, the double ppk/pstS mutant showed a higher production of the endogenous antibiotic actinorhodin and the heterologous antitumor 8-demethyl-tetracenomycin (up to 10-fold with respect to the wt strain), opening new possibilities for the use of this strain in the heterologous expression of antibiotic pathways.

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