Abstract OR-4: New Antibiotic Binding Site on the 30S Ribosomal Subunit

Background: Antibiotic resistance becomes one of the main problems of modern medicine; therefore, the development of new antibacterial compounds is absolutely necessary. The ribosome is the target for a lot of different antibiotics; there are several main binding sites on the ribosome – decoding center, peptidyl-transferase center, and ribosome exit tunnel. Modification or mutation of nucleotides in these sites could make cells resistant to structurally different antibiotics. Methods: pDualrep2 reporter system was used for detection of the protein synthesis inhibitors in cultural broths of new soil bacteria. By means of a cell-free translation system, the inhibitory activity and mechanism of action of Auraplanin were estimated. CryoEM data collection was performed on a Titan Krios operated at 300 kV, equipped with a Falcon II direct electron detector. Results: In this work, we have found a new inhibitor of protein synthesis, which binds in a completely new binding site. This compound is produced by Actinoplanes sp. VKM Ac-2862 and by Cryo-EM study of its complex with E.coli ribosome, it was shown, that it binds close to 560 loop of 30S ribosomal subunit. The new compound is a derivative of tetramic acid and we called it Auraplanin, because of bright orange color of the producer strain. Structural data are in good agreement with genetic results – resistant mutations were located close determined binding site. Substitutions C564G, G558U, and G566A significantly increase minimal inhibitory concentration, all these mutations were not detected previously. We also observed resistant mutation in ribosomal protein S4, this mutation was previously identified as error-prone. Interestingly, ribosomal ambiguity mutations, G299A and G347U, also increased resistance to Auraplanin. Conclusion: On the basis of the genetic, structural and biochemical studies we hypothesized that Auraplanin acts prevent the transfer from an open to a closed conformation of 30S subunit, in contrast to streptomycin, which promotes the formation of a closed state

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.Russian Foundation for Basic ResearchRevisión por pare

Mechanism of Translation Inhibition by Type II GNAT Toxin AtaT2

Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specifically target other if not all individual aminoacyl tRNAs

Bioprospecting for the soil-derived actinobacteria and bioactive secondary metabolites on the Western Qinghai-Tibet Plateau

IntroductionThe increase in incidence of multidrug-resistant bacteria and the inadequacy of new antimicrobial drugs have led to a widespread outbreak of bacterial antimicrobial resistance. To discover new antibiotics, biodiversity, and novelty of culturable actinobacteria dwelled in soil of the Western Qinghai-Tibet Plateau were investigated. By integrating antibacterial assay with omics tools, Amycolatopsis sp. A133, a rare actinobacterial strain and its secondary metabolites were further studied.MethodCulture-dependent method was used to obtain actinobacterial strains from two soil samples collected from Ali region in Qinghai-Tibet Plateau. The cultural extractions of representative strains were assayed against “ESKAPE” pathogens by paper-disk diffusion method and the double fluorescent protein reporter “pDualrep2” system. An Amycolatopsis strain coded as A133 was prioritized and its secondary metabolites were further analyzed and annotated by omics tools including antiSMASH and GNPS (Global Natural Social Molecular Networking). The predicted rifamycin analogs produced by Amycolatopsis sp. A133 were isolated and identified by chromatographic separation, such as Sephadex LH-20 and HPLC, and spectral analysis, such as NMR and UPLC-HRESI-MS/MS, respectively.ResultsA total of 406 actinobacteria strains affiliated to 36 genera in 17 families of 9 orders were isolated. Out of 152 representative strains, 63 isolates exhibited antagonistic activity against at least one of the tested pathogens. Among them, 7 positive strains were identified by the “pDualrep2” system as either an inhibitor of protein translation or DNA biosynthesis. The cultural broth of Amycolatopsis sp. A133 exhibited a broader antimicrobial activity and can induce expression of TurboRFP. The secondary metabolites produced by strain A133 was annotated as rifamycins and zampanolides by antiSMASH and GNPS analysis. Five members of rifamycins, including rifamycin W, protorifamycin I, rifamycin W-M1, proansamycin B, and rifamycin S, were purified and identified. Rifamycin W-M1, was found as a new member of the naturally occurring rifamycin group of antibiotics.DiscussionAssisted by omics tools, the successful and highly efficient discovery of rifamycins, a group of clinically used antibiotics from actinobacteria in Ali area encouraged us to devote more energy to explore new antibiotics from the soils on the Western Tibetan Plateau

Exploring the Diversity and Antibacterial Potentiality of Cultivable Actinobacteria from the Soil of the Saxaul Forest in Southern Gobi Desert in Mongolia

Saxaul (Haloxylon ammodendron) is the most widespread plant community in the Gobi Desert in Mongolia, which plays important roles in wind control, sand fixation and water conservation. Investigations of soil-derived actinobacteria inhabiting in the saxaul forest in Gobi Desert in Mongolia have been scarce. In this study, biodiversity of culturable actinobacteria isolated from soil of the saxaul forest in Southern Gobi Aimak (Southern Gobi Province) of Mongolia was characterized and their potential to produce compounds with antibacterial activities was assessed. A total of 172 actinobacterial strains were recovered by culture-based approaches and were phylogenetically affiliated into 22 genera in 13 families of seven orders. Forty-nine actinobacterial isolates were selected to evaluate the antibacterial activities and their underlying mechanism of action was screened by means of a dual-fluorescent reporter assay (pDualrep2). Twenty-three isolates exhibited antagonistic activity against at least one of the tested pathogens, of which two Streptomyces strains can attenuate protein translation by ribosome stalling. Combinational strategies based on modern metabolomics, including bioassay-guided thin-layer chromatography (TLC), UPLC-QTOF-MS/MS based structural annotation and enhanced molecular networking successfully annotated chloramphenicol, althiomycin and granaticin and their derivatives as the antibacterial compounds from extracts in three Streptomyces strains, respectively. This work demonstrates that UPLC-MS/MS-based structural identification and enhanced molecular networking are effective strategies to rapidly illuminate the bioactive chemicals in the microbial extracts. Meanwhile, our results show that the saxaul forest in Mongolia Gobi Desert is a prospective source for discovering novel actinobacteria and biologically active compounds

Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C

Microcin C (McC) is a peptide-nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects-ribosome induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori

Nanopore Sequencing for De Novo Bacterial Genome Assembly and Search for Single-Nucleotide Polymorphism

Nanopore sequencing (ONT) is a new and rapidly developing method for determining nucleotide sequences in DNA and RNA. It serves the ability to obtain long reads of thousands of nucleotides without assembly and amplification during sequencing compared to next-generation sequencing. Nanopore sequencing can help for determination of genetic changes leading to antibiotics resistance. This study presents the application of ONT technology in the assembly of an E. coli genome characterized by a deletion of the tolC gene and known single-nucleotide variations leading to antibiotic resistance, in the absence of a reference genome. We performed benchmark studies to determine minimum coverage depth to obtain a complete genome, depending on the quality of the ONT data. A comparison of existing programs was carried out. It was shown that the Flye program demonstrates plausible assembly results relative to others (Shasta, Canu, and Necat). The required coverage depth for successful assembly strongly depends on the size of reads. When using high-quality samples with an average read length of 8 Kbp or more, the coverage depth of 30× is sufficient to assemble the complete genome de novo and reliably determine single-nucleotide variations in it. For samples with shorter reads with mean lengths of 2 Kbp, a higher coverage depth of 50× is required. Avoiding of mechanical mixing is obligatory for samples preparation. Nanopore sequencing can be used alone to determine antibiotics-resistant genetic features of bacterial strains

Diversity, Novelty, and Antimicrobial Activity of Endophytic Actinobacteria From Mangrove Plants in Beilun Estuary National Nature Reserve of Guangxi, China

Endophytic actinobacteria are one of the important pharmaceutical resources and well known for producing different types of bioactive substances. Nevertheless, detection of the novelty, diversity, and bioactivity on endophytic actinobacteria isolated from mangrove plants are scarce. In this study, five different mangrove plants, Avicennia marina, Aegiceras corniculatum, Kandelia obovota, Bruguiera gymnorrhiza, and Thespesia populnea, were collected from Beilun Estuary National Nature Reserve in Guangxi Zhuang Autonomous Region, China. A total of 101 endophytic actinobacteria strains were recovered by culture-based approaches. They distributed in 7 orders, 15 families, and 28 genera including Streptomyces, Curtobacterium, Mycobacterium, Micrococcus, Brevibacterium, Kocuria, Nocardioides, Kineococcus, Kytococcus, Marmoricola, Microbacterium, Micromonospora, Actinoplanes, Agrococcus, Amnibacterium, Brachybacterium, Citricoccus, Dermacoccus, Glutamicibacter, Gordonia, Isoptericola, Janibacter, Leucobacter, Nocardia, Nocardiopsis, Pseudokineococcus, Sanguibacter, and Verrucosispora. Among them, seven strains were potentially new species of genera Nocardioides, Streptomyces, Amnibacterium, Marmoricola, and Mycobacterium. Above all, strain 8BXZ-J1 has already been characterized as a new species of the genus Marmoricola. A total of 63 out of 101 strains were chosen to screen antibacterial activities by paper-disk diffusion method and inhibitors of ribosome and DNA biosynthesis by means of a double fluorescent protein reporter. A total of 31 strains exhibited positive results in at least one antibacterial assay. Notably, strain 8BXZ-J1 and three other potential novel species, 7BMP-1, 5BQP-J3, and 1BXZ-J1, all showed antibacterial bioactivity. In addition, 21 strains showed inhibitory activities against at least one “ESKAPE” resistant pathogens. We also found that Streptomyces strains 2BBP-J2 and 1BBP-1 produce bioactive compound with inhibitory activity on protein biosynthesis as result of translation stalling. Meanwhile, Streptomyces strain 3BQP-1 produces bioactive compound inducing SOS-response due to DNA damage. In conclusion, this study proved mangrove plants harbored a high diversity of cultivable endophytic actinobacteria, which can be a promising source for discovery of novel species and bioactive compounds

RtcB2-PrfH Operon Protects E. coli ATCC25922 Strain from Colicin E3 Toxin

In the bid to survive and thrive in an environmental setting, bacterial species constantly interact and compete for resources and space in the microbial ecosystem. Thus, they have adapted to use various antibiotics and toxins to fight their rivals. Simultaneously, they have evolved an ability to withstand weapons that are directed against them. Several bacteria harbor colicinogenic plasmids which encode toxins that impair the translational apparatus. One of them, colicin E3 ribotoxin, mediates cleavage of the 16S rRNA in the decoding center of the ribosome. In order to thrive upon deployment of such ribotoxins, competing bacteria may have evolved counter-conflict mechanisms to prevent their demise. A recent study demonstrated the role of PrfH and the RtcB2 module in rescuing a damaged ribosome and the subsequent re-ligation of the cleaved 16S rRNA by colicin E3 in vitro. The rtcB2-prfH genes coexist as gene neighbors in an operon that is sporadically spread among different bacteria. In the current study, we report that the RtcB2-PrfH module confers resistance to colicin E3 toxicity in E. coli ATCC25922 cells in vivo. We demonstrated that the viability of E. coli ATCC25922 strain that is devoid of rtcB2 and prfH genes is impaired upon action of colicin E3, in contrast to the parental strain which has intact rtcB2 and prfH genes. Complementation of the rtcB2 and prfH gene knockout with a high copy number-plasmid (encoding either rtcB2 alone or both rtcB2-prfH operon) restored resistance to colicin E3. These results highlight a counter-conflict system that may have evolved to thwart colicin E3 activity

A New Albomycin-Producing Strain of <i>Streptomyces globisporus</i> subsp. <i>globisporus</i> May Provide Protection for Ants <i>Messor structor</i>

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