Mónica G. Malmierca

The genus Streptomyces is one of the largest producers of secondary metabolites with bioactive properties of interest. However, many of the genes involved in synthesizing these compounds are silenced under laboratory conditions. One of the strategies used to activate these metabolic pathways is the elimination of repressor genes, which prevent the transcription of other genes. In this work, the lsr2 gene has been selected for study since it is a repressor with a preference for binding to AT-rich regions, which makes it exert its effect especially on those horizontally transferred gene sequences that have a very different GC content to the core Streptomyces spp. genome. Therefore, the effects of the deletion of this gene are observed, and, in addition, a mapping of the potential binding sites of Lsr2 in Streptomyces spp. genomes is proposed. As a result of this gene knockout, the production of various secondary metabolites is overproduced and/or activated, which suggests that the study of this regulator can be interesting in the field of natural product discovery.

Background: Volatile compounds are key elements in the interaction and communication between organisms at both interspecific and intraspecific levels. In complex bacterial communities, the emission of these fast-acting chemical messengers allows an exchange of information even at a certain distance that can cause different types of responses in the receiving organisms. The changes in secondary metabolism as a consequence of this interaction arouse great interest in the field of searching for bioactive compounds since they can be used as a tool to activate silenced metabolic pathways. Regarding the great metabolic potential that the Actinobacteria group presents in the production of compounds with attractive properties, we evaluated the reply the emitted volatile compounds can generate in other individuals of the same group. Results: We recently reported that volatile compounds released by different streptomycete species trigger the modulation of biosynthetic gene clusters in Streptomyces spp. which finally leads to the activation/repression of the production of secondary metabolites in the recipient strains. Here we present the application of this rationale in a broader bacterial community to evaluate volatiles as signaling effectors that drive the activation of biosynthesis of bioactive compounds in other members of the Actinobacteria group. Using cocultures of different actinobacteria (where only the volatile compounds reach the recipient strain) we were able to modify the bacterial secondary metabolism that drives overproduction (e.g., granaticins, actiphenol, chromomycins) and/or de novo production (e.g., collismycins, skyllamycins, cosmomycins) of compounds belonging to different chemical species that present important biological activities. Conclusions: This work shows how the secondary metabolism of different Actinobacteria species can vary significantly when exposed in co-culture to the volatile compounds of other phylum-shared bacteria, these effects being variable depending on strains and culture media. This approach can be applied to the field of new drug discovery to increase the battery of bioactive compounds produced by bacteria that can potentially be used in treatments for humans and animals.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house “Carlos Sialer (CS)” strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide–depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.

The study of volatile organic compounds (VOCs) has expanded because of the growing need to search for new bioactive compounds that could be used as therapeutic alternatives. These small molecules serve as signals to establish interactions with other nearby organisms in the environment. In this work, we evaluated the antifungal effect of VOCs produced by different Streptomyces spp. This study was performed using VOC chamber devices that allow for the free exchange of VOCs without physical contact between microorganisms or the diffusible compounds they produce. Antifungal activity was tested against Escovopsis weberi, a fungal pathogen that affects ant nest stability, and the results showed that Streptomyces spp. CS014, CS057, CS131, CS147, CS159, CS207, and CS227 inhibit or reduce the fungal growth with their emitted VOCs. A GS-MS analysis of volatiles produced and captured by activated charcoal suggested that these Streptomyces strains synthesize several antifungal VOCs, many of them produced because of the presence of E. weberi, with the accumulation of various VOCs determining the growth inhibition effect.

The increasing appearance of multiresistant pathogens, as well as emerging diseases, has highlighted the need for new strategies to discover natural compounds that can be used as therapeutic alternatives, especially in the genus Streptomyces, which is one of the largest producers of bioactive metabolites. In recent years, the study of volatile compounds (VOCs) has raised interest because of the variety of their biological properties in addition to their involvement in cell communication. In this work, we analyze the implications of VOCs as mediating molecules capable of inducing the activation of biosynthetic pathways of bioactive compounds in surrounding Actinomycetes. For this purpose, several strains of Streptomyces were co-cultured in chamber devices that allowed VOC exchange while avoiding physical contact. In several of those strains, secondary metabolism was activated by VOCs emitted by companion strains, resulting in increased antibiotic production and synthesis of new VOCs. This study shows a novel strategy to exploit the metabolic potential of Actinomycetes as well as emphasizes the importance of studying the interactions between different microorganisms sharing the same ecological niche.