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There are several well-studied examples of protective symbiosis between insect host and symbiotic actinobacteria, producing antimicrobial metabolites to inhibit host pathogens. These mutualistic relationships are best described for some wasps and leaf-cutting ants, while a huge variety of insect species still remain poorly explored. For the first time, we isolated actinobacteria from the harvester ant Messor structor and evaluated the isolates’ potential as antimicrobial producers. All isolates could be divided into two morphotypes of single and mycelial cells. We found that the most common mycelial morphotype was observed among soldiers and least common among larvae in the studied laboratory colony. The representative of this morphotype was identified as Streptomyces globisporus subsp. globisporus 4-3 by a polyphasic approach. It was established using a E. coli JW5503 pDualRep2 system that crude broths of mycelial isolates inhibited protein synthesis in reporter strains, but it did not disrupt the in vitro synthesis of proteins in cell-free extracts. An active compound was extracted, purified and identified as albomycin δ2. The pronounced ability of albomycin to inhibit the growth of entomopathogens suggests that Streptomyces globisporus subsp. globisporus may be involved in defensive symbiosis with the Messor structor ant against infections.

Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants. Luminescence is engineered in whole plants, without an exogenous substrate, using a fungal gene cluster.

Marine polychaetes Odontosyllis undecimdonta, commonly known as fireworms, emit bright blue-green bioluminescence. Until the recent identification of the Odontosyllis luciferase enzyme, little progress had been made toward characterizing the key components of this bioluminescence system. Here we present the biomolecular mechanisms of enzymatic (leading to light emission) and nonenzymatic (dark) oxidation pathways of newly described O. undecimdonta luciferin. Spectral studies, including 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction, of isolated substances allowed us to characterize the luciferin as an unusual tricyclic sulfur-containing heterocycle. Odontosyllis luciferin does not share structural similarity with any other known luciferins. The structures of the Odontosyllis bioluminescent system’s low molecular weight components have enabled us to propose chemical transformation pathways for the enzymatic and nonspecific oxidation of luciferin.

The transition to organic farming is one of the most desirable achievements of our time. Rational use of organic farming approaches not only enables a reduction in costs and increased yields but also limits the risks associated with the use of pesticides and chemicals. Despite the widest practical application of numerous biocontrol agents based on strains, their metabolome, including the main active substances, often remains unknown. In order to understand the basic principles of the functioning of the K-3618 strain, widely used in organic farming, we studied its spectrum of antimicrobial metabolites in detail. It was shown that the main antimicrobial agents of K-3618 are representatives of the macrolactin family. The identified macrolactin A (MLN A) and its acylated analogs 7-O-malonyl macrolactin A (mal-MLN A) and 7-O-succinyl macrolactin A (suc-MLN A) are active against Gram-positive bacterial pathogens, including multidrug-resistant strains. Among them, suc-MLN A is the most potent antimicrobial, highly active (MIC = 0.1 μg/mL) against the common human pathogen methicillin-resistant (MRSA). It was revealed that the primary mechanism of action of MLN A-based macrolactins is protein translation inhibition. Acylated macrolactins outperform MLN A in the prokaryotic cell-free system, displaying high efficiency in low micromolar concentrations. We observed that acylated MLN A analogs undergo pathogen-mediated biotransformation into MLN F analogs, having their antimicrobial activity reduced by two orders of magnitude. Hence, both acylation of MLNs and stabilization of the MLN A core are essential for the creation of new synthetic MLNs with improved antimicrobial activity and stability. However, we speculate that these degradability modes are of prime importance for bacterial ecology, and they are highly conserved in species from various ecological niches.

Oxydifficidin is a natural polyketide antibiotic that has long been recognized as a ribosome-targeting agent that inhibits protein synthesis. In this paper, we describe strain EV17 and compare its complete genome sequence with that of the previously characterized strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry to elucidate the production of oxydifficidin by these strains. Toeprinting and small fluorescent peptide assays showed that isolated oxydifficidin induces a generalized inhibition of translation at every step of elongation in protein biosynthesis. In previous studies, it has been demonstrated that oxydifficidin targets bL12 protein. Although spontaneous mutations conferring resistance to oxydifficidin in ribosomal protein bL12 located relatively close to the thiostrepton binding site on uL11, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We are the first to show that this compound does not affect eukaryotic translation and has two orders of magnitude lower effect on eukaryotic cells compared to bacteria. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic agent.

Puromycin (Puro) is a natural aminonucleoside antibiotic that inhibits protein synthesis by its incorporation into elongating peptide chains. The unique mechanism of Puro finds diverse applications in molecular biology, including the selection of genetically engineered cell lines, in situ protein synthesis monitoring, and studying ribosome functions. However, the key step of Puro biosynthesis remains enigmatic. In this work, pur6-guided genome mining is carried out to explore the natural diversity of Puro-like antibiotics. The diversity of biosynthetic gene cluster (BGC) architectures suggests the existence of distinct structural analogs of puromycin encoded by pur-like clusters. Moreover, the presence of tRNACys in some BGCs, i.e., cst-like clusters, leads us to the hypothesis that Pur6 utilizes aminoacylated tRNA as an activated peptidyl precursor, resulting in cysteine-based analogs. Detailed metabolomic analysis of Streptomyces sp. VKM Ac-502 containing cst-like BGC revealed the production of a cysteinyl-based analog of Puro—cystocin (Cst). Similar to puromycin, cystocin inhibits both prokaryotic and eukaryotic translation by the same mechanism. Aminonucleoside N-acetyltransferase CstC inactivated Cst, mediating antibiotic resistance in genetically modified bacteria and human cells. The substrate specificity of CstC originated from the steric hindrance of its active site. We believe that novel aminonucleosides and their inactivating enzymes can be developed through the directed evolution of the discovered biosynthetic machinery.

Biochemistry of bioluminescence of the marine parchment tubeworm has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from algae, which demonstrate bioluminescence activity with luciferase in the presence of Fe ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.

Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fe[sup.2+] ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.