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.

The untapped potential of actinobacterial lanthipeptides as therapeutic agents.

The increase in bacterial resistance generated by the indiscriminate use of antibiotics in medical practice set new challenges for discovering bioactive natural products as alternatives for therapeutics. Lanthipeptides are an attractive natural product group that has been only partially explored and shows engaging biological activities. These molecules are small peptides with potential application as therapeutic agents. Some members show antibiotic activity against problematic drug-resistant pathogens and against a wide variety of viruses. Nevertheless, their biological activities are not restricted to antimicrobials, as their contribution to the treatment of cystic fibrosis, cancer, pain symptoms, control of inflammation, and blood pressure has been demonstrated. The study of biosynthetic gene clusters through genome mining has contributed to accelerating the discovery, enlargement, and diversification of this group of natural products. In this review, we provide insight into the recent advances in the development and research of actinobacterial lanthipeptides that hold great potential as therapeutics.

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.

The Use of Particulate Systems for Tuberculosis Prophylaxis and Treatment: Opportunities and Challenges.

The use of particles to develop vaccines and treatments for a wide variety of diseases has increased, and their success has been demonstrated in preclinical investigations. Accurately targeting cells and minimizing doses and adverse side effects, while inducing an adequate biological response, are important advantages that particulate systems offer. The most used particulate systems are liposomes and their derivatives, immunostimulatory complexes, virus-like particles, and organic or inorganic nano- and microparticles. Most of these systems have been proven using therapeutic or prophylactic approaches to control tuberculosis, one of the most important infectious diseases worldwide. This article reviews the progress and current state of the use of particles for the administration of TB vaccines and treatments in vitro and in vivo, with a special emphasis on polymeric particles. In addition, we discuss the challenges and benefits of using these particulate systems to provide researchers with an overview of the most promising strategies in current preclinical trials, offering a perspective on their progress to clinical trials.

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.

The Role of Mucoadhesion and Mucopenetration in the Immune Response Induced by Polymer-Based Mucosal Adjuvants.

Mucus is a viscoelastic gel that acts as a protective barrier for epithelial surfaces. The mucosal vehicles and adjuvants need to pass through the mucus layer to make drugs and vaccine delivery by mucosal routes possible. The mucoadhesion of polymer particle adjuvants significantly increases the contact time between vaccine formulations and the mucosa; then, the particles can penetrate the mucus layer and epithelium to reach mucosa-associated lymphoid tissues. This review presents the key findings that have aided in understanding mucoadhesion and mucopenetration while exploring the influence of physicochemical characteristics on mucus-polymer interactions. We describe polymer-based particles designed with mucoadhesive or mucopenetrating properties and discuss the impact of mucoadhesive polymers on local and systemic immune responses after mucosal immunization. In future research, more attention paid to the design and development of mucosal adjuvants could lead to more effective vaccines.

In vivo tracing of immunostimulatory raw starch microparticles after mucosal administration.

Raw starch microparticles (SMPs) proved efficient antigen carriers with adjuvant properties when administered via the mucosal route; however, the underlying mechanisms associated with this bioactivity are unknown. In the present study, we explored the mucoadhesion properties, fate, and toxicity of starch microparticles after mucosal administration. Nasally administered microparticles were mainly retained in nasal turbinates, reaching the nasal-associated lymphoid tissue; this step is facilitated by the ability of the microparticles to penetrate through the mucous epithelium. Likewise, we found intraduodenally administered SMPs on the small intestinal villi, follicle-associated epithelium, and Peyer's patches. Furthermore, under simulated gastric and intestinal pH conditions, we detected mucoadhesion between the SMPs and mucins, regardless of microparticle swelling. SMPs' mucoadhesion and translocation to mucosal immune responses induction sites explain the previously reported role of these microparticles as vaccine adjuvants and immunostimulants.

Understanding the Phagocytosis of Particles: the Key for Rational Design of Vaccines and Therapeutics.

A robust comprehension of phagocytosis is crucial for understanding its importance in innate immunity. A detailed description of the molecular mechanisms that lead to the uptake and clearance of endogenous and exogenous particles has helped elucidate the role of phagocytosis in health and infectious or autoimmune diseases. Furthermore, knowledge about this cellular process is important for the rational design and development of particulate systems for the administration of vaccines or therapeutics. Depending on these specific applications and the required biological responses, particles must be designed to encourage or avoid their phagocytosis and prolong their circulation time. Functionalization with specific polymers or ligands and changes in the size, shape, or surface of particles have important effects on their recognition and internalization by professional and nonprofessional phagocytes and have a major influence on their fate and safety. Here, we review the phagocytosis of particles intended to be used as carrier or delivery systems for vaccines or therapeutics, the cells involved in this process depending on the route of administration, and the strategies employed to obtain the most desirable particles for each application through the manipulation of their physicochemical characteristics. We also offer a view of the challenges and potential opportunities in the field and give some recommendations that we expect will enable the development of improved approaches for the rational design of these systems.

Bioinformatic comparison of three Embleya species and description of steffimycins production by Embleya sp. NF3.

The Embleya genus is a new member of the Streptomycetaceae family formed by only two species isolated from soil (Embleya scabrispora and Embleya hyalina). Strain NF3 is an endophytic actinobacterium obtained from the medicinal tree Amphipterygium adstringens. By 16S rRNA gene analysis, NF3 strain was identified as Embleya sp., closely related to E. hyalina. In our interest to deep into the NF3 strain features, a bioinformatic study was performed on the Embleya genus based on their genome information to produce secondary metabolites. A comparative analysis of the biosynthetic gene clusters (BGCs) of NF3 with the two released Embleya genomes revealed that NF3 has 49 BGCs, E. scabrispora DSM41855 has 50 BGCs, and E. hyalina NBRC13850 has 46 BGCs. Although bearing similar cluster numbers, the three strains shared only 25% of the BGCs information. NF3 encoded the nybomycin cluster detected in E. hyalina NBRC13850 and lacked the hitachimycin cluster present in E. scabrispora DSM41855. On the contrary, strain NF3 contained a cluster for the anthracycline steffimycin, neither encoded by E. hyalina NBRC13850 nor by E. scabrispora DSM41855. Our results and previous characterization studies supported strain NF3 as a new member of the genus Embleya. The chemical analysis of the steffimycins produced by strain NF3 showed the production of eight compounds of the steffimycins and steffimycinone families. Four of these molecules have already been described: steffimycin B, steffimycin C, 8-demethoxy-10-deoxysteffimycinone, and 7-deoxiesteffimycinone, and four are new natural products: 8-demethoxysteffimycin B, 8-demethoxy-10-deoxysteffimycin B, 7-deoxy-8-demethoxysteffimycinone, and 7-deoxy-10-deoxysteffimycinone. With this information, we proposed an alternative pathway to produce StefB. Among steffimycins, StefB was the main compound produced by this strain (29.8%) and showed the best cytotoxic activity. KEY POINTS: • The Embleya genus and its biosynthetic potential • An alternative biosynthetic pathway for steffimycins biosynthesis • Four new natural products of the steffimycin family.

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.

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.

Evolutionary genomics and biosynthetic potential of novel environmental Actinobacteria.

Actinobacteria embroil Gram-positive microbes with high guanine and cytosine contents in their DNA. They are the source of most antimicrobials of bacterial origin utilized in medicine today. Their genomes are among the richest in novel secondary metabolites with high biotechnological potential. Actinobacteria reveal complex patterns of evolution, responses, and adaptations to their environment, which are not yet well understood. We analyzed three novel plant isolates and explored their habitat adaptation, evolutionary patterns, and potential secondary metabolite production. The phylogenomically characterized isolates belonged to Actinoplanes sp. TFC3, Streptomyces sp. L06, and Embleya sp. NF3. Positively selected genes, relevant in strain evolution, encoded enzymes for stress resistance in all strains, including porphyrin, chlorophyll, and ubiquinone biosynthesis in Embleya sp. NF3. Streptomyces sp. L06 encoded for pantothenate and proteins for CoA biosynthesis with evidence of positive selection; furthermore, Actinoplanes sp. TFC3 encoded for a c-di-GMP synthetase, with adaptive mutations. Notably, the genomes harbored many genes involved in the biosynthesis of at least ten novel secondary metabolites, with many avenues for future new bioactive compound characterization-specifically, Streptomyces sp. L06 could make new ribosomally synthesized and post-translationally modified peptides, while Embleya sp. NF3 could produce new non-ribosomal peptide synthetases and ribosomally synthesized and post-translationally modified peptides. At the same time, TFC3 has particularly enriched in terpene and polyketide synthases. All the strains harbored conserved genes in response to diverse environmental stresses, plant growth promotion factors, and degradation of various carbohydrates, which supported their endophytic lifestyle and showed their capacity to colonize other niches. This study aims to provide a comprehensive estimation of the genomic features of novel Actinobacteria. It sets the groundwork for future research into experimental tests with new bioactive metabolites with potential application in medicine, biofertilizers, and plant biomass residue utilization, with potential application in medicine, as biofertilizers and in plant biomass residues utilization. KEY POINTS: • Potential of novel environmental bacteria for secondary metabolites production • Exploring the genomes of three novel endophytes isolated from a medicinal tree • Pan-genome analysis of Actinobacteria genera.

Metaproteomic Insights Into the Microbial Community in Pozol.

Pozol is an acidic, refreshing, and non-alcoholic traditional Mayan beverage made with nixtamalized corn dough that is fermented spontaneously. The extensive analysis of the microbiology, biochemistry and metaproteomics of pozol allowed the construction of a comprehensive image of the fermentation system. The main changes in both the substrate and the microbiota occurred in the first 9 h of fermentation. The increase in microorganisms correlated with the drop in pH and with the decrease in the contents of carbohydrates, lipids, and nitrogen, which shows that this stage has the highest metabolic activity. Bacterial proteins were mainly represented by those of lactic acid bacteria, and among them, the proteins from genus Streptococcus was overwhelmingly the most abundant. Yeast proteins were present in all the analyzed samples, while proteins from filamentous fungi increased up to 48 h. The metaproteomic approach allowed us to identify several previously unknown enzyme complexes in the system. Additionally, enzymes for hydrolysis of starch, hemicellulose and cellulose were found, indicating that all these substrates can be used as a carbon source by the microbiota. Finally, enzymes related to the production of essential intermediates involved in the synthesis of organic acids, acetoin, butanediol, fatty acids and amino acids important for the generation of compounds that contribute to the sensorial quality of pozol, were found.

Nitrogen Fixation in Pozol, a Traditional Fermented Beverage.

Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed.IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

Tolerance to acid and alkali by Streptococcus infantarius subsp. infantarius strain 25124 isolated from fermented nixtamal dough: Pozol. Studies in APT broth.

Pozol is a beverage prepared with maize dough made after boiling the kernels in limewater. This pretreatment could act as a selective force that shapes the starter microbiota, with microorganisms able to survive the fermentation. Since Streptococcus infantarius subsp. infantarius (Sii) dominates in pozol, we evaluated the effect of acid and alkali stresses on strain Sii-25124 in commercial APT broth as a first attempt to assess its adaptation capacity. Results suggest that Sii-25124 has adaptative advantages to pH changes that possibly contribute to its persistence even after the acidification of the dough. Its cardinal pH values were 4.0 and 11.0, with an optimum between 6.6 and 8.0. It showed alkali tolerance unlike other pozol Sii strains. Adaptation at pH 4.0, 10.0 and 11.0, compared with non-adapted cells, induced acid tolerance enhancing survival at pH 3.6 (P < 0.05); a 2 min heat shock at 62 °C induced alkali tolerance response enhancing survival at pH 10.5 (P < 0.05). The up-regulation of dnaK, groEL, ptsG and atpB was observed during 5 h of exposition at pH 3.6, 4.0 and 10.0, showing similar expression rates after induction by acid shock or alkaline stress. Changes of atpB were more evident having almost five-fold induction during long-term stress.

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.

Omics in traditional vegetable fermented foods and beverages.

For a long time, food microbiota has been studied using traditional microbiological techniques. With the arrival of molecular or culture-independent techniques, a strong understanding of microbiota dynamics has been achieved. However, analyzing the functional role of microbial communities is not an easy task. The application of omics sciences to the study of fermented foods would provide the metabolic and functional understanding of the microbial communities and their impact on the fermented product, including the molecules that define its aroma and flavor, as well as its nutritional properties. Until now, most omics studies have focused on commercial fermented products, such as cheese, wine, bread and beer, but traditional fermented foods have been neglected. Therefore, the information that allows to relate the present microbiota in the food and its properties remains limited. In this review, reports on the applications of omics in the study of traditional fermented foods and beverages are reviewed to propose new ways to analyze the fermentation phenomena.

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.

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.

The structure of (E)-biformene synthase provides insights into the biosynthesis of bacterial bicyclic labdane-related diterpenoids.

The labdane-related diterpenoids (LRDs) are a large group of natural products with a broad range of biological activities. They are synthesized through two consecutive reactions catalyzed by class II and I diterpene synthases (DTSs). The structural complexity of LRDs mainly depends on the catalytic activity of class I DTSs, which catalyze the formation of bicyclic to pentacyclic LRDs, using as a substrate the catalytic product of class II DTSs. To date, the structural and mechanistic details for the biosynthesis of bicyclic LRDs skeletons catalyzed by class I DTSs remain unclear. This work presents the first X-ray crystal structure of an (E)-biformene synthase, LrdC, from the soil bacterium Streptomyces sp. strain K155. LrdC was identified as a part of an LRD cluster of five genes and was found to be a class I DTS that catalyzes the Mg2+-dependent synthesis of bicyclic LRD (E)-biformene by the dephosphorylation and rearrangement of normal copalyl pyrophosphate (CPP). Structural analysis of LrdC coupled with docking studies suggests that Phe189 prevents cyclization beyond the bicyclic LRD product through a strong stabilization of the allylic carbocation intermediate, while Tyr317 functions as a general base catalyst to deprotonate the CPP substrate. Structural comparisons of LrdC with homology models of bacterial bicyclic LRD-forming enzymes (CldD, RmnD and SclSS), as well as with the crystallographic structure of bacterial tetracyclic LRD ent-kaurene synthase (BjKS), provide further structural insights into the biosynthesis of bacterial LRD natural products.