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Organic Letters Aug 2021Zwitterionic carbohydrate modifications, such as phosphoethanolamine (PEtN), govern host-pathogen interactions. Whereas it is recognized that these modifications...
Zwitterionic carbohydrate modifications, such as phosphoethanolamine (PEtN), govern host-pathogen interactions. Whereas it is recognized that these modifications stimulate the host immune system, the purpose of PEtN modification remains largely descriptive. As an enabling step toward studying this carbohydrate modification, we report a synthesis of the zwitterionic trisaccharide repeating unit. The 32-step synthesis was enabled by H-phosphonate chemistry to install the PEtN arm on a poorly reactive and sterically hindered C4-alcohol.
Topics: Ethanolamines; Molecular Structure; Photorhabdus; Trisaccharides
PubMed: 34314177
DOI: 10.1021/acs.orglett.1c02023 -
Nucleic Acids Research Aug 2021Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to...
Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.
Topics: ADP-Ribosylation; Bacterial Toxins; GTP Phosphohydrolases; NAD; Peptide Elongation Factor G; Photorhabdus; Protein Biosynthesis; RNA, Ribosomal, 23S; Thiostrepton; Type VI Secretion Systems
PubMed: 34255843
DOI: 10.1093/nar/gkab608 -
PeerJ 2021Biological control using entomopathogenic nematodes (EPN) has demonstrated good potential to contribute to the integral control of mosquito larvae, which as adults are...
BACKGROUND
Biological control using entomopathogenic nematodes (EPN) has demonstrated good potential to contribute to the integral control of mosquito larvae, which as adults are vectors of diseases such as Dengue fever, Zika and Chikungunya. However, until now there are no records of the presence of EPN or their killing capacity in Yucatán state, southern México. The objectives of the current study were: (1) to report the entomopathogenic nematodes present in Yucatán soils and (2) to determine the killing capacity of the most frequent and abundant EPN against mosquito larvae and the microbial community developed by exposed to this EPN.
METHODS
The nematodes were collected by the insect trap technique using the great wax moth . Internal transcribed spacer (ITS), 28S gene of ribosomal DNA and phylogenetic analyses were performed to identify the EPN. For the bioassay, four concentrations of the most frequent and abundant EPN were tested: 1,260:1 infective juveniles (IJs) per mosquito larvae, 2,520 IJs:1, 3,780 IJs:1 and 5,040 IJs:1. High-throughput sequencing of the 16S rRNA gene was used to identify bacterial amplicon sequences in the mosquito larvae infected with EPN.
RESULTS
Six isolates of were recovered from 144 soil samples. (four isolates) was the most frequent and abundant EPN, followed by n. sp. (two isolates). Both nematodes are reported for the first time for Yucatán state, Mexico. The concentration of 2,520 IJs:1 produced 80% of mosquito larvae mortality in 48 h. Representative members of genus were numerically dominant (74%) in mosquito larvae infected by . It is most likely that these bacteria produce secondary toxic metabolites that enhance the mortality of these mosquito larvae.
PubMed: 34249499
DOI: 10.7717/peerj.11633 -
Gene Aug 2021The genome sequences of entomopathogenic nematode (EPN) bacteria and their functional analyses can lead to the genetic engineering of the bacteria for use as biocontrol...
Genome assembly and annotation of Photorhabdus heterorhabditis strain ETL reveals genetic features involved in pathogenicity with its associated entomopathogenic nematode and anti-host effectors with biocontrol potential applications.
The genome sequences of entomopathogenic nematode (EPN) bacteria and their functional analyses can lead to the genetic engineering of the bacteria for use as biocontrol agents. The bacterial symbiont Photorhabdus heterorhabditis strain ETL isolated from an insect pathogenic nematode, Heterorhabditis zealandica strain ETL, collected in the northernmost region of South Africa was studied to reveal information that can be useful in the design of improvement strategies for both effective and liquid production method of EPN-based pesticides. The strain ETL genome was found closely related to the type strain genome of P. australis DSM 17,609 (~60 to 99.9% CDSs similarity), but closely related to the not yet genome-sequenced type strain, P. heterorhabditis. It has a genome size of 4,866,148 bp and G + C content of 42.4% similar to other Photorhabdus. It contains 4,351 protein coding genes (CDSs) of which, at least 84% are shared with the de facto type strain P. luminescens subsp. laumondii TTO1, and has 318 unknown CDSs and the genome has a higher degree of plasticity allowing it to adapt to different environmental conditions, and to be virulent against various insects; observed through genes acquired through horizontal gene transfer mechanisms, clustered regularly interspaced short palindromic repeats, non-determined polyketide- and non-ribosomal peptide- synthase gene clusters, and many genes associated with uncharacterized proteins; which also justify the strain ETL's genes differences (quantity and quality) compared to P. luminescens subsp. laumondii TTO1. The protein coding sequences contained genes with both bio-engineering and EPNs mass production importance, of which numerous are uncharacterized.
Topics: Animals; Base Sequence; Biological Control Agents; Genes, Bacterial; Genome, Bacterial; Host-Pathogen Interactions; Molecular Sequence Annotation; Photorhabdus; Phylogeny; Strongyloidea; Virulence
PubMed: 34147570
DOI: 10.1016/j.gene.2021.145780 -
Biomolecules May 2021A broad spectrum of volatile organic compounds' (VOCs') biological activities has attracted significant scientific interest, but their mechanisms of action remain little...
A broad spectrum of volatile organic compounds' (VOCs') biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCs-the cyclic monoterpenes (-)-limonene and (+)-α-pinene-on bacteria was studied in this work. We used genetically engineered bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the genes of We showed that (-)-limonene induces the P and P promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (-)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-α-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (-)-limonene completely inhibits the DnaKJE-ClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both wild type and mutant Δ strains. (+)-α-Pinene partially inhibits refolding only in Δ mutant strain.
Topics: Bacterial Proteins; Bicyclic Monoterpenes; DNA Damage; DNA, Bacterial; Escherichia coli K12; Limonene; Photorhabdus; SOS Response, Genetics
PubMed: 34072355
DOI: 10.3390/biom11060806 -
Biosensors May 2021LuxR and LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of genes in the presence of an autoinducer...
LuxR and LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the cells, genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. -based bacterial -biosensors were used, in which genes were controlled by LuxR-dependent promoters and , , or regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30-100 pM).
Topics: 4-Butyrolactone; Acyl-Butyrolactones; Aliivibrio; Biosensing Techniques; Escherichia coli; Genes, Regulator; Promoter Regions, Genetic; Trans-Activators
PubMed: 34071046
DOI: 10.3390/bios11060166 -
Insects May 2021The meadow spittlebug (Hemiptera: Aphrophoridae) is the primary vector of (Proteobacteria: Xanthomonadaceae) in Europe, a pest-disease complex of economically relevant...
The meadow spittlebug (Hemiptera: Aphrophoridae) is the primary vector of (Proteobacteria: Xanthomonadaceae) in Europe, a pest-disease complex of economically relevant crops such as olives, almonds, and grapevine, managed mainly through the use of broad-spectrum pesticides. Providing environmentally sound alternatives to reduce the reliance on chemical control is a primary challenge in the control of and, hence, in the protection of crops against the expansion of its associated bacterial pathogen. Entomopathogenic nematodes (EPNs) are well-known biocontrol agents of soil-dwelling arthropods. Recent technological advances in field applications, including improvements in obtaining cell-free supernatant from their symbiotic bacteria, allow their successful implementation against aerial pests. Thus, this study aimed to evaluate, for the first time, the efficacy of EPN applications against nymphal instars of We tested four EPN species and the cell-free supernatant of their corresponding symbiotic bacteria: -, -, -, and - subsp. . First, we showed that 24 and 72 h exposure to the foam produced by nymphs did not affect virulence. The direct application of steinernematid EPNs provided promising results, reaching 90, 78, and 53% nymphal mortality rates after five days of exposure for , , and , respectively. Conversely, the application of the cell-free supernatant from resulted in nymphal mortalities of 64%, significantly higher than observed for species after five days of exposure. Overall, we demonstrated the great potential of the application of specific EPNs and cell-free supernatant of their symbiont bacteria against , introducing new opportunities to develop them as biopesticides for integrated management practices or organic vineyard production.
PubMed: 34068952
DOI: 10.3390/insects12050448 -
Angewandte Chemie (International Ed. in... Aug 2021Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Efficient engineering of these often...
Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge. Here we describe a cloning and co-expression strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 220 mg L . Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains. These synthetic type of NRPS (type S) enables easier access to engineering, overcomes cloning limitations, and provides a simple and rapid approach to building peptide libraries via the combination of different NRPS subunits.
Topics: Escherichia coli; Leucine Zippers; Peptide Synthases; Peptides; Photorhabdus; Plasmids; Proof of Concept Study; Protein Engineering; Xenorhabdus
PubMed: 34015175
DOI: 10.1002/anie.202102859 -
Molecular Plant-microbe Interactions :... Sep 2021High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a strain chromosomally tagged with the Nanoluc...
High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a strain chromosomally tagged with the Nanoluc luciferase (NL) from the deep-sea shrimp , a bioluminescent marker significantly brighter than the conventional firefly luciferase. Our reporter strain tagged with NL was more than 100 times brighter than tagged with the operon from , one of the existing luciferase-based strains. In planta imaging was improved by using the surfactant Silwet L-77, particularly at a lower reporter concentration. Using this imaging system, more than 30 epigenetic mutants were analyzed for their resistance traits because the defense signaling pathway is known to be epigenetically regulated. SWC1, a defense-related chromatin remodeling complex, was found to be a positive defense regulator, which supported one of two earlier conflicting reports. Compromises in DNA methylation in the CG context led to enhanced resistance against virulent pv. . Dicer-like and Argonaute proteins, important in the biogenesis and exerting the effector function of small RNAs, respectively, showed modest but distinct requirements for effector-triggered immunity and basal resistance to pv. . In addition, the transcriptional expression of an epigenetic component was found to be a significant predictor of its immunity contribution. In summary, this study showcased how a high-throughput resistance assay enabled by a pathogen strain with an improved luminescent reporter could provide insightful knowledge about complex defense signaling pathways.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Topics: Arabidopsis; Arabidopsis Proteins; Epigenesis, Genetic; Luciferases; Luminescence; Plant Diseases; Pseudomonas syringae; Signal Transduction
PubMed: 34010013
DOI: 10.1094/MPMI-12-20-0351-TA -
Environmental Microbiology Reports Oct 2021The resistance-nodulation-division (RND)-type efflux pumps AcrAB and MdtABC contribute to multidrug-resistance (MDR) in Gram-negative bacteria. Photorhabdus is a...
The resistance-nodulation-division (RND)-type efflux pumps AcrAB and MdtABC contribute to multidrug-resistance (MDR) in Gram-negative bacteria. Photorhabdus is a symbiotic bacterium of soil nematodes that also produces virulence factors killing insects by septicaemia. We previously showed that mdtA deletion in Photorhabdus laumondii TT01 resulted in no detrimental phenotypes. Here, we investigated the roles of the last two putative RND transporters in TT01 genome, AcrAB and AcrAB-like (Plu0759-Plu0758). Only ΔacrA and ΔmdtAΔacrA mutants were multidrug sensitive, even to triphenyltetrazolium chloride and bromothymol blue used for Photorhabdus isolation from nematodes on the nutrient bromothymol blue-triphenyltetrazolium chloride agar (NBTA) medium. Both mutants also displayed slightly attenuated virulence after injection into Spodoptera littoralis. Transcriptional analysis revealed intermediate levels of acrAB expression in vitro, in vivo and post-mortem, whereas its putative transcriptional repressor acrR was weakly expressed. Yet, plasmid-mediated acrR overexpression did not decrease acrAB transcript levels neither MDR in TT01 WT. While no pertinent mutations were detected in acrR of the same P. laumondii strain grown either on NBTA or nutrient agar, we suggest that AcrR-mediated repression of acrAB is not physiologically required under conditions tested. Finally, we propose that AcrAB is the primary RND-efflux pump, which is essential for MDR in Photorhabdus and may confer adaptive advantages during insect infection.
Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Insecta; Photorhabdus; Virulence
PubMed: 34002534
DOI: 10.1111/1758-2229.12974