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Nucleic Acids Research Mar 2015Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be...
Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed 'recombineering', in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5'-3' exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.
Topics: Amino Acid Sequence; Bacterial Proteins; Bacteriophage lambda; DNA, Bacterial; Escherichia coli; Exodeoxyribonuclease V; Genetic Engineering; Genome, Bacterial; Genomics; Molecular Sequence Data; Multigene Family; Operon; Photorhabdus; Plasmids; Recombination, Genetic; Sequence Homology, Amino Acid; Xenorhabdus
PubMed: 25539914
DOI: 10.1093/nar/gku1336 -
Frontiers in Microbiology 2019A major issue currently facing medicine is antibiotic resistance. No new class of antibiotics for the treatment of Gram-negative infections has been introduced in more... (Review)
Review
A major issue currently facing medicine is antibiotic resistance. No new class of antibiotics for the treatment of Gram-negative infections has been introduced in more than 40 years. We screened a collection of and strains in the quest to discover new structures that are active against the most problematic multidrug-resistant bacteria. These species are symbiotic bacteria of entomopathogenic nematodes and their life cycle, the richness of the bacteria's genome in non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, and their propensity to produce secondary metabolites with a large diversity of chemical structures make them a good starting point to begin an ambitious drug discovery program. Odilorhabdins (ODLs), a novel antibacterial class, were identified from this campaign. These compounds inhibit bacterial translation by binding to the small ribosomal subunit at a site not exploited by current antibiotics. Following the development of the total synthesis of this family of peptides, a medicinal chemistry program was started to optimize their pharmacological properties. NOSO-502, the first ODL preclinical candidate was selected. This compound is currently under preclinical development for the treatment of multidrug-resistant Gram-negative infections in hospitalized patients.
PubMed: 31921069
DOI: 10.3389/fmicb.2019.02893 -
Proceedings of the National Academy of... Jan 2015It is well recognized that bacteria communicate via small diffusible molecules, a process termed quorum sensing. The best understood quorum sensing systems are those...
It is well recognized that bacteria communicate via small diffusible molecules, a process termed quorum sensing. The best understood quorum sensing systems are those that use acylated homoserine lactones (AHLs) for communication. The prototype of those systems consists of a LuxI-like AHL synthase and a cognate LuxR receptor that detects the signal. However, many proteobacteria possess LuxR receptors, yet lack any LuxI-type synthase, and thus these receptors are referred to as LuxR orphans or solos. In addition to the well-known AHLs, little is known about the signaling molecules that are sensed by LuxR solos. Here, we describe a novel cell-cell communication system in the insect and human pathogen Photorhabdus asymbiotica. We identified the LuxR homolog PauR to sense dialkylresorcinols (DARs) and cyclohexanediones (CHDs) instead of AHLs as signals. The DarABC synthesis pathway produces the molecules, and the entire system emerged as important for virulence. Moreover, we have analyzed more than 90 different Photorhabdus strains by HPLC/MS and showed that these DARs and CHDs are specific to the human pathogen P. asymbiotica. On the basis of genomic evidence, 116 other bacterial species are putative DAR producers, among them many human pathogens. Therefore, we discuss the possibility of DARs as novel and widespread bacterial signaling molecules and show that bacterial cell-cell communication goes far beyond AHL signaling in nature.
Topics: Acyl-Butyrolactones; Amino Acid Sequence; Animals; Bacterial Proteins; Base Sequence; Conserved Sequence; Cyclohexanones; DNA, Bacterial; Genes, Bacterial; Humans; Models, Biological; Models, Molecular; Molecular Sequence Data; Operon; Photorhabdus; Phylogeny; Protein Conformation; Quorum Sensing; Repressor Proteins; Resorcinols; Sequence Homology, Amino Acid; Signal Transduction; Trans-Activators
PubMed: 25550519
DOI: 10.1073/pnas.1417685112 -
Frontiers in Microbiology 2023Contractile injection systems (CISs) are phage tail-related structures that are encoded in many bacterial genomes. These devices encompass the cell-based type VI... (Review)
Review
Contractile injection systems (CISs) are phage tail-related structures that are encoded in many bacterial genomes. These devices encompass the cell-based type VI secretion systems (T6SSs) as well as extracellular CISs (eCISs). The eCISs comprise the R-tailocins produced by various bacterial species as well as related phage tail-like structures such as the antifeeding prophages (Afps) of , the virulence cassettes (PVCs), and the metamorphosis-associated contractile structures (MACs) of . These contractile structures are released into the extracellular environment upon suicidal lysis of the producer cell and play important roles in bacterial ecology and evolution. In this review, we specifically portray the eCISs with a focus on the R-tailocins, sketch the history of their discovery and provide insights into their evolution within the bacterial host, their structures and how they are assembled and released. We then highlight ecological and evolutionary roles of eCISs and conceptualize how they can influence and shape bacterial communities. Finally, we point to their potential for biotechnological applications in medicine and agriculture.
PubMed: 37886057
DOI: 10.3389/fmicb.2023.1264877 -
Biology Nov 2022Entomopathogenic nematodes (EPNs) are insect parasitic nematodes of the genera and . These nematodes are symbiotically associated with the bacteria, and respectively....
Entomopathogenic nematodes (EPNs) are insect parasitic nematodes of the genera and . These nematodes are symbiotically associated with the bacteria, and respectively. National parks in Thailand are a potentially rich resource for recovering native EPNs and their symbiotic bacteria. The objectives of this study are to isolate and identify EPNs and their bacterial flora from soil samples in four national parks in Thailand and to evaluate their efficacy for controlling mosquito larvae. Using a baiting method with a moth larvae and a White trap technique, 80 out of 840 soil samples (9.5%) from 168 field sites were positive for EPNs. Sequencing of an internal transcribed spacer resulted in the molecular identification of nematode isolates as , and SGmg3, while using 28S rDNA sequencing, nematode species were identified as , , , and one closely related to . For the symbiotic bacterial isolates, based on sequencing, the spp. were identified as subsp. , subsp. and subsp. . isolates were identified as , , , and . Results of bioassays demonstrate that isolates were effective on both and . Therefore, we conclude that soil from Thailand's national parks contain a high diversity of entomopathogenic nematodes and their symbiotic bacteria. bacteria are larvicidal against culicine mosquitoes and may serve as effective biocontrol agents.
PubMed: 36421372
DOI: 10.3390/biology11111658 -
Nature Communications Jun 2020Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction...
Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.
Topics: Animals; Bacterial Toxins; Binding Sites; Cell Adhesion; Cell Membrane; HEK293 Cells; Heparin; Humans; Insecta; Lewis X Antigen; Models, Molecular; Molecular Docking Simulation; Morganella morganii; Photorhabdus; Polysaccharides; Xenorhabdus
PubMed: 32483155
DOI: 10.1038/s41467-020-16536-7 -
Journal of Fungi (Basel, Switzerland) Apr 2022is a phytopathogenic fungus that causes devastating losses in strawberries without effective countermeasures. Members of the genus exhibit antimicrobial capability and...
is a phytopathogenic fungus that causes devastating losses in strawberries without effective countermeasures. Members of the genus exhibit antimicrobial capability and have been found to have the potential for use as biocontrol agents against . species exhibit two phase variations with a differentiated composition of secondary metabolites designated to each phase. In this study, sp. nov. 0813-124 exhibited phase I (PL1) and phase II (PL2); however, only PL1 displayed distinct inhibition of in the confrontation assay. We identified the bioactive ingredients of sp. nov. 0813-124 to be glidobactin A and cepafungin I, with MIC values lower than 1.5 and 2.0 µg/mL, respectively. Furthermore, we revealed the biosynthetic gene cluster (BGC) of corresponding bioactive molecules through genomics analysis and determined its expression level in PL1 and PL2. The expression of glidobactin BGC in PL1 increased rapidly within 24 h, while PL2 was eventually stimulated after 60 h. In summary, we demonstrated that sp. nov. 0813-124 could potentially be used as a biocontrol agent or part of a natural product repertoire for combating .
PubMed: 35448634
DOI: 10.3390/jof8040403 -
Scientific Reports Nov 2023The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study,...
The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.
Topics: Genome, Bacterial; Bacteria; Computational Biology; Multigene Family; Biosynthetic Pathways
PubMed: 38007490
DOI: 10.1038/s41598-023-47121-9 -
Analytical Chemistry Dec 2019Whole-cell and cell-free transcription-translation biosensors have recently become favorable alternatives to conventional detection methods, as they are cost-effective,...
Whole-cell and cell-free transcription-translation biosensors have recently become favorable alternatives to conventional detection methods, as they are cost-effective, environmental friendly, and easy to use. Importantly, the biological responses from the biosensors need to be converted into a physicochemical signal for easy detection, and a variety of genetic reporters have been employed for this purpose. Reporter gene selection is vital to a sensor performance and application success. However, it was largely based on trial and error with very few systematic side-by-side investigations reported. To address this bottleneck, here we compared eight reporters from three reporter categories, i.e., fluorescent (, , , ), colorimetric (), and bioluminescent ( from and , ) reporters, under the control of two representative biosensors for mercury- and quorum-sensing molecules. Both whole-cell and cell-free formats were investigated to assess key sensing features including limit of detection (LOD), input and output dynamic ranges, response time, and output visibility. For both whole-cell biosensors, the lowest detectable concentration of analytes and the fastest responses were achieved with NanoLuc. Notably, we developed, to date, the most sensitive whole-cell mercury biosensor using NanoLuc as reporter, with an LOD ≤ 50.0 fM HgCl 30 min postinduction. For cell-free biosensors, overall, and led to shorter response time and lower LOD than the others. This comprehensive profile of diverse reporters in a single setting provides a new important benchmark for reporter selection, aiding the rapid development of whole-cell and cell-free biosensors for various applications in the environment and health.
Topics: Aliivibrio fischeri; Biosensing Techniques; Escherichia coli; Genes, Reporter; Mercury; Photorhabdus; Quorum Sensing
PubMed: 31690077
DOI: 10.1021/acs.analchem.9b04444 -
Applied and Environmental Microbiology Aug 2020The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease...
The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease resistance and abiotic stress tolerance in crops. Here, we investigated the capacity of the secondary variant to react to plant root exudates and their behavior toward microorganisms in the rhizosphere. is known to live in symbiosis with entomopathogenic nematodes (EPNs) and to be highly pathogenic toward insects. The -EPN relationship has been widely studied, and this combination has been used as a biological control agent; however, not much attention has been paid to the putative lifestyle of in the rhizosphere. We performed transcriptome analysis to show how responds to plant root exudates. The analysis highlighted genes involved in chitin degradation, biofilm regulation, formation of flagella, and type VI secretion system. Furthermore, we provide evidence that can inhibit growth of phytopathogenic fungi. Finally, we demonstrated a specific interaction of with plant roots. Understanding the role and the function of this bacterium in the rhizosphere might accelerate the progress in biocontrol manipulation and elucidate the peculiar mechanisms adopted by plant growth-promoting rhizobacteria in plant root interactions. Insect-pathogenic bacteria are widely used in biocontrol strategies against pests. Very little is known about the life of these bacteria in the rhizosphere. Here, we show that can specifically react to and interact with plant roots. Understanding the adaptation of in the rhizosphere is highly important for the biotechnological application of entomopathogenic bacteria and could improve future sustainable pest management in agriculture.
Topics: Biological Control Agents; Chemotaxis; Exudates and Transudates; Fungi; Gene Expression Profiling; Genes, Bacterial; Photorhabdus; Plant Roots; RNA-Seq; Rhizosphere
PubMed: 32591378
DOI: 10.1128/AEM.00891-20