An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Trends in the two-component system's role in the synthesis of antibiotics by Streptomyces.

Despite the advances in understanding the regulatory networks for secondary metabolite production in Streptomyces, the participation of the two-component systems (TCS) in this process still requires better characterization. These sensing systems and their responses to environmental stimuli have been described by evaluating mutant strains with techniques that allow in-depth regulatory responses. However, defining the stimulus that triggers their activation is still a task. The transmembrane nature of the sensor kinases and the high content of GC in the streptomycetes represent significant challenges in their study. In some examples, adding elements to the assay medium has determined the respective ligand. However, a complete TCS description and characterization requires specific amounts of the involved proteins that are most difficult to obtain. The availability of enough sensor histidine kinase concentrations could facilitate the identification of the ligand-protein interaction, and besides would allow the establishment of its phosphorylation mechanisms and determine their tridimensional structure. Similarly, the advances in the development of bioinformatics tools and novel experimental techniques also promise to accelerate the TCSs description and provide knowledge on their participation in the regulation processes of secondary metabolite formation. This review aims to summarize the recent advances in the study of TCSs involved in antibiotic biosynthesis and to discuss alternatives to continue their characterization. KEY POINTS: • TCSs are the environmental signal transducers more abundant in nature. • The Streptomyces have some of the highest number of TCSs found in bacteria. • The study of signal transduction between SHKs and RRs domains is a big challenge.

Opportunities and challenges of microbial siderophores in the medical field.

Siderophores are low-molecular-weight secondary metabolites that function as iron chelators. Under iron-deficiency conditions, they are produced by a wide variety of microbes, allowing them to increase their iron uptake. The primary function of these compounds is the environmental iron scavenging and its transport into the cytosol. Iron is then reduced to its ferrous form to operate as an enzymatic cofactor for various functions, including respiration, nitrogen fixation, photosynthesis, methanogenesis, and amino acid synthesis. Depending on their functional group, siderophores are classified into hydroxamate, catecholate, phenolate, carboxylate, and mixed types. They have achieved great importance in recent years due to their medical applications as antimicrobial, antimalarial, or anticancer drugs, vaccines, and drug-delivery agents. This review integrates current advances in specific healthcare applications of microbial siderophores, delineating new opportunities and challenges as viable therapies to fight against diseases that represent crucial public health problems in the medical field.Key points• Siderophores are low-molecular-weight secondary metabolites functioning as iron chelators.• The siderophore's properties offer viable options to face diverse clinical problems.• Siderophores are alternatives for the enhancement of antibiotic activities.

Impact of novel microbial secondary metabolites on the pharma industry.

Microorganisms are remarkable producers of a wide diversity of natural products that significantly improve human health and well-being. Currently, these natural products comprise half of all the pharmaceuticals on the market. After the discovery of penicillin by Alexander Fleming 85 years ago, the search for and study of antibiotics began to gain relevance as drugs. Since then, antibiotics have played a valuable role in treating infectious diseases and have saved many human lives. New molecules with anticancer, hypocholesterolemic, and immunosuppressive activity have now been introduced to treat other relevant diseases. Smaller biotechnology companies and academic laboratories generate novel antibiotics and other secondary metabolites that big pharmaceutical companies no longer develop. The purpose of this review is to illustrate some of the recent developments and to show the potential that some modern technologies like metagenomics and genome mining offer for the discovery and development of new molecules, with different functions like therapeutic alternatives needed to overcome current severe problems, such as the SARS-CoV-2 pandemic, antibiotic resistance, and other emerging diseases. KEY POINTS: • Novel alternatives for the treatment of infections caused by bacteria, fungi, and viruses. • Second wave of efforts of microbial origin against SARS-CoV-2 and related variants. • Microbial drugs used in clinical practice as hypocholesterolemic agents, immunosuppressants, and anticancer therapy.

Correction to: Deletion of the hypothetical protein SCO2127 of Streptomyces coelicolor allowed identification of a new regulator of actinorhodin production.

The published online version of this paper contains mistake. The authors first and last names have been interchanged. The correct version is given above.

Production of distinct labdane-type diterpenoids using a novel cryptic labdane-like cluster from Streptomyces thermocarboxydus K155.

Bioinformatic mining of the Streptomyces thermocarboxydus K155 genome predicted the presence of four synthases for the production of geosmin, hopene, albaflavenone, and a type B-type A diterpenoid system like that described for labdane-related diterpenoids (LRD). The lrd cluster was comprised by an operon of four genes (lrdABDC). This cluster seemed to be silent in the wild-type strain, as neither labdane nor terpene-like compounds were detected by UPLC-TOF-MS and GC-MS analyses in both culture supernatants and mycelial extracts. Heterologous expression of the lrdABDC cluster in a defective cyslabdan producer (Streptomyces cyslabdanicus K04-0144Δcld) generated 8,17-epoxy-7-hydroxy labda-12,14-diene and cyslabdan. The same cluster expressed in the strains Streptomyces coelicolor M1152, Streptomyces peucetius var. caesius, and Streptomyces avermitilis SUKA22 produced the general intermediary labda-8(17), 12(E),14-triene [(E)-biformene]. Besides (E)-biformene, S. coelicolor M1152 and S. avermitilis SUKA22 produced two and three different labdane-type diterpenoids, underlying the relevance of the genetic background of the Streptomyces host in product formation.

Interplay between carbon, nitrogen and phosphate utilization in the control of secondary metabolite production in Streptomyces.

Streptomyces species are a wide and diverse source of many therapeutic agents (antimicrobials, antineoplastic and antioxidants, to name a few) and represent an important source of compounds with potential applications in medicine. The effect of nitrogen, phosphate and carbon on the production of secondary metabolites has long been observed, but it was not until recently that the molecular mechanisms on which these effects rely were ascertained. In addition to the specific macronutrient regulatory mechanisms, there is a complex network of interactions between these mechanisms influencing secondary metabolism. In this article, we review the recent advances in our understanding of the molecular mechanisms of regulation exerted by nitrogen, phosphate and carbon sources, as well as the effects of their interconnections, on the synthesis of secondary metabolites by members of the genus Streptomyces.

Carbon catabolite regulation in Streptomyces: new insights and lessons learned.

One of the most significant control mechanisms of the physiological processes in the genus Streptomyces is carbon catabolite repression (CCR). This mechanism controls the expression of genes involved in the uptake and utilization of alternative carbon sources in Streptomyces and is mostly independent of the phosphoenolpyruvate phosphotransferase system (PTS). CCR also affects morphological differentiation and the synthesis of secondary metabolites, although not all secondary metabolite genes are equally sensitive to the control by the carbon source. Even when the outcome effect of CCR in bacteria is the same, their essential mechanisms can be rather different. Although usually, glucose elicits this phenomenon, other rapidly metabolized carbon sources can also cause CCR. Multiple efforts have been put through to the understanding of the mechanism of CCR in this genus. However, a reasonable mechanism to explain the nature of this process in Streptomyces does not yet exist. Several examples of primary and secondary metabolites subject to CCR will be examined in this review. Additionally, recent advances in the metabolites and protein factors involved in the Streptomyces CCR, as well as their mechanisms will be described and discussed in this review.

Transcriptomic analysis of a classical model of carbon catabolite regulation in Streptomyces coelicolor.

BACKGROUND: In the genus Streptomyces, one of the most remarkable control mechanisms of physiological processes is carbon catabolite repression (CCR). This mechanism regulates the expression of genes involved in the uptake and utilization of alternative carbon sources. CCR also affects the synthesis of secondary metabolites and morphological differentiation. Even when the outcome effect of CCR in different bacteria is the same, their essential mechanisms can be quite different. In several streptomycetes glucose kinase (Glk) represents the main glucose phosphorylating enzyme and has been regarded as a regulatory protein in CCR. To evaluate the paradigmatic model proposed for CCR in Streptomyces, a high-density microarray approach was applied to Streptomyces coelicolor M145, under repressed and non-repressed conditions. The transcriptomic study was extended to assess the ScGlk role in this model by comparing the transcriptomic profile of S. coelicolor M145 with that of a ∆glk mutant derived from the wild-type strain, complemented with a heterologous glk gene from Zymomonas mobilis (Zmglk), insensitive to CCR but able to grow in glucose (ScoZm strain). RESULTS: Microarray experiments revealed that glucose influenced the expression of 651 genes. Interestingly, even when the ScGlk protein does not have DNA binding domains and the glycolytic flux was restored by a heterologous glucokinase, the ScGlk replacement modified the expression of 134 genes. From these, 91 were also affected by glucose while 43 appeared to be under the control of ScGlk. This work identified the expression of S. coelicolor genes involved in primary metabolism that were influenced by glucose and/or ScGlk. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. CONCLUSIONS: The transcriptome of a classical model of CCR was studied in S. coelicolor to differentiate between the effects due to glucose or ScGlk in this regulatory mechanism. Glucose elicited important metabolic and transcriptional changes in this microorganism. While its entry and flow through glycolysis and pentose phosphate pathway were stimulated, the gluconeogenesis was inhibited. Glucose also triggered the CCR by repressing transporter systems and the transcription of enzymes required for secondary carbon sources utilization. Our results confirm and update the agar model of the CCR in Streptomyces and its dependence on the ScGlk per se. Surprisingly, the expected regulatory function of ScGlk was not found to be as global as thought before (only 43 out of 779 genes were affected), although may be accompanied or coordinated by other transcriptional regulators. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. These findings offer new opportunities to study and understand the CCR in S. coelicolor by increasing the number of known glucose and ScGlk -regulated pathways and a new set of putative regulatory proteins possibly involved or controlling the CCR.

Deletion of the hypothetical protein SCO2127 of Streptomyces coelicolor allowed identification of a new regulator of actinorhodin production.

Although the specific function of SCO2127 remains elusive, it has been assumed that this hypothetical protein plays an important role in carbon catabolite regulation and therefore in antibiotic biosynthesis in Streptomyces coelicolor. To shed light on the functional relationship of SCO2127 to the biosynthesis of actinorhodin, a detailed analysis of the proteins differentially produced between the strain M145 and the Δsco2127 mutant of S. coelicolor was performed. The delayed morphological differentiation and impaired production of actinorhodin showed by the deletion strain were accompanied by increased abundance of gluconeogenic enzymes, as well as downregulation of both glycolysis and acetyl-CoA carboxylase. Repression of mycothiol biosynthetic enzymes was further observed in the absence of SCO2127, in addition to upregulation of hydroxyectoine biosynthetic enzymes and SCO0204, which controls nitrite formation. The data generated in this study reveal that the response regulator SCO0204 greatly contributes to prevent the formation of actinorhodin in the ∆sco2127 mutant, likely through the activation of some proteins associated with oxidative stress that include the nitrite producer SCO0216.

Biochemistry and regulatory functions of bacterial glucose kinases.

Glucokinases (Glks) are enzymes widely distributed in all three domains of life. They are located at the beginning of the glycolytic pathway and are responsible for the glucose phosphorylation from various phosphate group donors such as ATP, ADP and polyphosphate. So far, there are eight crystallized Glks, and at least one belongs to each of the three reported Glk families. Structural studies have elucidated the mechanism for Glk action and multimerization. Cloning, overexpression and biochemical characterization have demonstrated the wide diversity of these enzymes. As reported for various microorganisms, in addition to their catalytic activity, some Glks, possessing ROK (Repressor Orf Kinases) motifs, also display a regulatory role. This function has been associated to the mechanisms of carbon catabolite regulation, morphological differentiation and antibiotic production. The present review covers the classification, detailed tertiary structure, mechanism of action, biochemical characterization and some regulatory aspects of bacterial Glks.

Glucose kinases from Streptomyces peucetius var. caesius.

Glucose kinases (Glks) are enzymes of the glycolytic pathway involved in glucose phosphorylation. These enzymes can use various phosphoryl donors such as ATP, ADP, and polyphosphate. In several streptomycetes, ATP-glucose kinase (ATP-Glk) has been widely studied and regarded as the main glucose phosphorylating enzyme and is likely a regulatory protein in carbon catabolite repression. In cell extracts from the doxorubicin overproducing strain Streptomyces peucetius var. caesius, grown in glucose, a polyphosphate-dependent Glk (Pp-Glk) was detected by zymogram. Maximum activity was observed during the stationary growth phase (48 h) of cells grown in 100 mM glucose. No activity was detected when 20 mM glutamate was used as the only carbon source, supporting a role for glucose in inducing this enzyme. Contrary to wild-type strains of Streptomyces coelicolor, Streptomyces lividans, and Streptomyces thermocarboxydus K-155, S. peucetius var. caesius produced 1.8 times more Pp-Glk than ATP-Glk. In addition, this microorganism produced five and four times more Pp-Glk and anthracyclines, respectively, than its wild-type S. peucetius parent strain, supporting a role for this enzyme in antibiotic production in the overproducer strain. A cloned 726-bp DNA fragment from S. peucetius var. caesius encoded a putative Pp-Glk, with amino acid identities between 83 and 87 % to orthologous sequences from the above-cited streptomycetes. The cloned fragment showed the polyphosphate-binding sequences GXDIGGXXIK, TXGTGIGSA, and KEX(4)SWXXWA. Sequences for the Zn-binding motif were not detected in this fragment, suggesting that Pp-Glk is not related to the Glk ROK family of proteins.

Targeting the Impossible: A Review of New Strategies against Endospores.

Endospore-forming bacteria are ubiquitous, and their endospores can be present in food, in domestic animals, and on contaminated surfaces. Many spore-forming bacteria have been used in biotechnological applications, while others are human pathogens responsible for a wide range of critical clinical infections. Due to their resistant properties, it is challenging to eliminate spores and avoid the reactivation of latent spores that may lead to active infections. Furthermore, endospores play an essential role in the survival, transmission, and pathogenesis of some harmful strains that put human and animal health at risk. Thus, different methods have been applied for their eradication. Nevertheless, natural products are still a significant source for discovering and developing new antibiotics. Moreover, targeting the spore for clinical pathogens such as Clostridioides difficile is essential to disease prevention and therapeutics. These strategies could directly aim at the structural components of the spore or their germination process. This work summarizes the current advances in upcoming strategies and the development of natural products against endospores. This review also intends to highlight future perspectives in research and applications.

Carbon catabolite regulation of secondary metabolite formation, an old but not well-established regulatory system.

Secondary microbial metabolites have various functions for the producer microorganisms, which allow them to interact and survive in adverse environments. In addition to these functions, other biological activities may have clinical relevance, as diverse as antimicrobial, anticancer and hypocholesterolaemic effects. These metabolites are usually formed during the idiophase of growth and have a wide diversity in their chemical structures. Their synthesis is under the impact of the type and concentration of the culture media nutrients. Some of the molecular mechanisms that affect the synthesis of secondary metabolites in bacteria (Gram-positive and negative) and fungi are partially known. Moreover, all microorganisms have their peculiarities in the control mechanisms of carbon sources, even those belonging to the same genus. This regulatory knowledge is necessary to establish culture conditions and manipulation methods for genetic improvement and product fermentation. As the carbon source is one of the essential nutritional factors for antibiotic production, its study has been imperative both at the industrial and research levels. This review aims to draw the utmost recent advances performed to clarify the molecular mechanisms of the negative effect exerted by the carbon source on the secondary metabolite formation, emphasizing those found in Streptomyces, one of the genera most profitable antibiotic producers.

Recent findings of molecules with anti-infective activity: screening of non-conventional sources.

In recent years, the number of pathogenic microorganisms resistant to antibiotics has increased alarmingly. For the next 10-20 years, health organizations forecast high human mortality caused by these microorganisms. Therefore, the search for new anti-infectives is quite necessary and urgent. Traditionally, antibiotic-producing microorganisms have been isolated from common soil samples. However, this source seems to be exhausted considering the very few examples of antibiotic-producing microorganisms reported recently. In this review, non-conventional sources of anti-infective producing microorganisms are presented as a possible way to look for new and more effective compounds. These sources included arid soils, caves, areas with high temperatures (hot springs), high salinity or oceans and seas. Finally, other non-conventional sources of antibiotics reviewed are animal and invertebrate venoms, among others.

Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain.

Bacterial conjugation is a powerful tool used for DNA transfer from Escherichia coli into various bacteria including streptomycetes. In this methodology, spores are usually employed as recipient cells of the genetic information. However, some industrially important Streptomyces do not produce spores making difficult their genetic manipulation. In these strains, the use of mechanically fragmented mycelia allows DNA transfer with low efficiency. Streptomyces peucetius var. caesius is a non-sporulating bacteria which produces the antitumor compound doxorubicin. The use of aerial mycelia of this microorganism, failed to get intergeneric conjugation with E. coli. In the present work, by using young aerial mycelia of this microorganism and an excess of E. coli cells (~7×108cellsmL-1) in soybean-mannitol medium (MS) supplemented with 20mMMgCl2 resulted in a high number of exconjugant colonies (5×10-4) when compared to other reports from this genus (1.1×10-5 to 2.5×10-8). The effectiveness of these conditions was confirmed by isolating null mutants of two different glucokinases from S. peucetius var. caesius. The novelty in using young aerial mycelia as receptor cells, allowed an efficient conjugative process and opened the way for genetic manipulation of additional non-spore forming actinobacteria exhibiting natural resistance to be genetically manipulated.