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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 -
Biology Feb 2022(Lepidoptera: Lycaenidae) and (Lepidoptera: Pyralidae) are the key pests of pomegranates in Saudi Arabia that are managed mainly using broad-spectrum pesticides....
Isolation, Identification, and Biocontrol Potential of Entomopathogenic Nematodes and Associated Bacteria against (Lepidoptera: Lycaenidae) and (Lepidoptera: Pyralidae).
(Lepidoptera: Lycaenidae) and (Lepidoptera: Pyralidae) are the key pests of pomegranates in Saudi Arabia that are managed mainly using broad-spectrum pesticides. Interactions between the entomopathogenic nematodes (EPNs) Steinernematids, and Heterorhabditids, and their entomopathogenic bacterial symbionts (EPBs) have long been considered monoxenic 2-partner associations responsible for killing insects and, therefore, are widely used in insect pest biocontrol. However, there are limited reports identifying such organisms in Taif, Saudi Arabia. The current study aimed to identify the EPNs and their associated bacteria isolated from Taif, Saudi Arabia, and evaluate their biocontrol potential on third instar larvae of and under laboratory conditions. A total of 35 EPN isolates belonging to (20) and (15) were recovered from 320 soil samples. Twenty-six isolates of symbiotic or associated bacteria were isolated from EPNs and molecularly identified as (6 isolates), (4 isolates), (7), or (9). A pathogenicity assay revealed that spp. were more virulent than spp. against the two pomegranate insects, with LC values of 18.5 and 13.6 infective juveniles (IJs)/larva of for spp. and 52 and 32.4 IJs/larva of for spp. at 48 and 72 h post-treatment, respectively. Moreover, LC values of 9 and 6.6 IJs/larva ( spp.) and 34.4 and 26.6 IJs/larva ( spp.) were recorded for larvae at 48 and 72 h post-treatment. In addition, the EPB CQ1, isolated from spp., surpassed SJ10, associated with spp., in their ability to kill or larvae within 6 h post-application, resulting in 100% mortality in both insects after 24 and 48 h of exposure. We conclude that either application of EPNs' IJs or their associated EPBs could serve as potential biocontrol agents for and .
PubMed: 35205161
DOI: 10.3390/biology11020295 -
Frontiers in Cellular and Infection... 2014Bacteria communicate via small diffusible molecules to mediate group-coordinated behavior, a process designated as quorum sensing. The basic molecular quorum sensing...
Bacteria communicate via small diffusible molecules to mediate group-coordinated behavior, a process designated as quorum sensing. The basic molecular quorum sensing system of Gram-negative bacteria consists of a LuxI-type autoinducer synthase producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor detecting the AHLs to control expression of specific genes. However, many proteobacteria possess one or more unpaired LuxR-type receptors that lack a cognate LuxI-like synthase, referred to as LuxR solos. The enteric and insect pathogenic bacteria of the genus Photorhabdus harbor an extraordinarily high number of LuxR solos, more than any other known bacteria, and all lack a LuxI-like synthase. Here, we focus on the presence and the different types of LuxR solos in the three known Photorhabdus species using bioinformatics analyses. Generally, the N-terminal signal-binding domain (SBD) of LuxR-type receptors sensing AHLs have a motif of six conserved amino acids that is important for binding and specificity of the signaling molecule. However, this motif is altered in the majority of the Photorhabdus-specific LuxR solos, suggesting the use of other signaling molecules than AHLs. Furthermore, all Photorhabdus species contain at least one LuxR solo with an intact AHL-binding motif, which might allow the ability to sense AHLs of other bacteria. Moreover, all three species have high AHL-degrading activity caused by the presence of different AHL-lactonases and AHL-acylases, revealing a high quorum quenching activity against other bacteria. However, the majority of the other LuxR solos in Photorhabdus have a N-terminal so-called PAS4-domain instead of an AHL-binding domain, containing different amino acid motifs than the AHL-sensors, which potentially allows the recognition of a highly variable range of signaling molecules that can be sensed apart from AHLs. These PAS4-LuxR solos are proposed to be involved in host sensing, and therefore in inter-kingdom signaling. Overall, Photorhabdus species are perfect model organisms to study bacterial communication via LuxR solos and their role for a symbiotic and pathogenic life style.
Topics: Acyl-Butyrolactones; Amino Acid Motifs; Carboxylic Ester Hydrolases; Conserved Sequence; Genome, Bacterial; Photorhabdus; Phylogeny; Position-Specific Scoring Matrices; Protein Binding; Protein Interaction Domains and Motifs; Quorum Sensing; Repressor Proteins; Signal Transduction; Trans-Activators
PubMed: 25478328
DOI: 10.3389/fcimb.2014.00166 -
Insects Oct 2018causes considerable economic damage to small and thin-skinned fruits including cherry, blueberry, raspberry, grape and strawberry. Since it attacks fruits at the...
causes considerable economic damage to small and thin-skinned fruits including cherry, blueberry, raspberry, grape and strawberry. Since it attacks fruits at the ripening stage, the use of chemical pesticides is limited due to the high risk of residues on fruit. Biological control is thus expected to play an essential role in managing this pest. The Gram-negative bacterium, and its symbiotic spp. nematode have been shown to be highly pathogenic to insects, with a potential for replacing pesticides to suppress several pests. Insecticidal activity of at different bacterial cell concentrations and its cell-free supernatant were assessed against third-instar larvae and pupae of under laboratory conditions. suspensions had a significant oral and contact toxicity on larvae and pupae, with mortalities up to of 70⁻100% 10 days after treatment. Cell-free supernatant in the diet also doubled mortality rates of feeding larvae. Our results suggest that may be a promising candidate for biological control of , and its use in integrated pest management (IPM) programs is discussed.
PubMed: 30360545
DOI: 10.3390/insects9040148 -
PLoS Computational Biology Feb 2021The vast expansion of protein sequence databases provides an opportunity for new protein design approaches which seek to learn the sequence-function relationship...
The vast expansion of protein sequence databases provides an opportunity for new protein design approaches which seek to learn the sequence-function relationship directly from natural sequence variation. Deep generative models trained on protein sequence data have been shown to learn biologically meaningful representations helpful for a variety of downstream tasks, but their potential for direct use in the design of novel proteins remains largely unexplored. Here we show that variational autoencoders trained on a dataset of almost 70000 luciferase-like oxidoreductases can be used to generate novel, functional variants of the luxA bacterial luciferase. We propose separate VAE models to work with aligned sequence input (MSA VAE) and raw sequence input (AR-VAE), and offer evidence that while both are able to reproduce patterns of amino acid usage characteristic of the family, the MSA VAE is better able to capture long-distance dependencies reflecting the influence of 3D structure. To confirm the practical utility of the models, we used them to generate variants of luxA whose luminescence activity was validated experimentally. We further showed that conditional variants of both models could be used to increase the solubility of luxA without disrupting function. Altogether 6/12 of the variants generated using the unconditional AR-VAE and 9/11 generated using the unconditional MSA VAE retained measurable luminescence, together with all 23 of the less distant variants generated by conditional versions of the models; the most distant functional variant contained 35 differences relative to the nearest training set sequence. These results demonstrate the feasibility of using deep generative models to explore the space of possible protein sequences and generate useful variants, providing a method complementary to rational design and directed evolution approaches.
Topics: Algorithms; Computational Biology; Computer Simulation; Escherichia coli; Machine Learning; Neural Networks, Computer; Oxidoreductases; Photorhabdus; Proteins; Recombinant Proteins; Reproducibility of Results; Solubility
PubMed: 33635868
DOI: 10.1371/journal.pcbi.1008736 -
Insects Nov 2021The European grapevine moth (EGVM) (Lepidoptera: Tortricidae) is a relevant pest in the Palearctic region vineyards and is present in the Americas. Their management...
The European grapevine moth (EGVM) (Lepidoptera: Tortricidae) is a relevant pest in the Palearctic region vineyards and is present in the Americas. Their management using biological control agents and environmentally friendly biotechnical tools would reduce intensive pesticide use. The entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae are well-known virulent agents against arthropod pests thanks to symbiotic bacteria in the genera and (respectively) that produce natural products with insecticidal potential. Novel technological advances allow field applications of EPNs and those bioactive compounds as powerful bio-tools against aerial insect pests. This study aimed to determine the viability of four EPN species (, , , and ) as biological control agents against EGVM larval instars (L1, L3, and L5) and pupae. Additionally, the bioactive compounds from their four symbiotic bacteria (, , , and subsp. respectively) were tested as unfiltered ferment (UF) and cell-free supernatant (CFS) against the EGVM larval instars L1 and L3. All of the EPN species showed the capability of killing EGVM during the larval and pupal stages, particularly (mortalities of ~50% for L1 and >75% for L3 and L5 in only two days), followed by efficacy by . Similarly, the bacterial bioactive compounds produced higher larval mortality at three days against L1 (>90%) than L3 (~50%), making the application of UF more virulent than the application of CFS. Our findings indicate that both steinernematid species and their symbiotic bacterial bioactive compounds could be considered for a novel agro-technological approach to control in vineyards. Further research into co-formulation with adjuvants is required to expand their viability when implemented for aboveground grapevine application.
PubMed: 34821833
DOI: 10.3390/insects12111033 -
Marine Drugs Dec 2017In aquaculture, shrimp farming is a popular field. The benefits of shrimp farming include a relatively short grow-out time, high sale price, and good cost recovery.... (Review)
Review
In aquaculture, shrimp farming is a popular field. The benefits of shrimp farming include a relatively short grow-out time, high sale price, and good cost recovery. However, outbreaks of serious diseases inflict serious losses, and acute hepatopancreatic necrosis disease (AHPND) is an emerging challenge to this industry. In South American white shrimp () and grass shrimp (), this disease has a 70-100% mortality. The pathogenic agent of AHPND is a specific strain of which contains PirA and PirB toxins encoded in the pVA1 plasmid. PirA and PirB have been shown to cause the typical histological symptoms of AHPND in infected shrimps, and in this review, we will focus on our structural understanding of these toxins. By analyzing their structures, a possible cytotoxic mechanism, as well as strategies for anti-AHPND drug design, is proposed.
Topics: Animals; Aquaculture; Aquatic Organisms; Bacterial Proteins; Penaeidae; Toxins, Biological; Vibrio parahaemolyticus
PubMed: 29194352
DOI: 10.3390/md15120373 -
Infection and Immunity May 2018is an outstanding model for studying host antipathogen defense. Although substantial progress has been made in understanding how metabolism and immunity are...
is an outstanding model for studying host antipathogen defense. Although substantial progress has been made in understanding how metabolism and immunity are interrelated in flies, little information has been obtained on the molecular players that regulate metabolism and inflammation in during pathogenic infection. Recently, we reported that the inactivation of thioester-containing protein 2 () and promotes survival and decreases the bacterial burden in flies upon infection with the virulent pathogens and Here, we investigated physiological and pathological defects in mutant flies in response to challenge. We find that and loss-of-function mutant flies contain increased levels of carbohydrates and triglycerides in the presence or absence of infection. We also report that infection leads to higher levels of nitric oxide and reduced transcript levels of the apical caspase-encoding gene in and mutants. We show that and are upregulated mainly in the fat body rather than the gut in -infected wild-type flies and that mutants contain decreased numbers of bacteria in both tissue types. We propose that the inactivation of or in adult flies results in lower levels of inflammation and increased energy reserves in response to , which could confer a survival-protective effect during the initial hours of infection.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Host-Pathogen Interactions; Immunity, Innate; Inflammation
PubMed: 29463615
DOI: 10.1128/IAI.00810-17 -
Advanced Science (Weinheim,... Jun 2024Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii...
Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold. Here, multiple crystal structures of PlAntI in various conformations and with bound substrate surrogates or inhibitors are reported. Structure-based mutagenesis and activity assays provide experimental insights into the three sequential reaction steps to yield the natural product AQ-256. For comparison, a series of ligand-complex structures of two functionally related hydrolases involved in the biosynthesis of 1,8-dihydroxynaphthalene-melanin in pathogenic fungi is determined. These data provide fundamental insights into the mechanism of polyketide trimming that shapes pigments in pro- and eukaryotes.
Topics: Anthraquinones; Polyketides; Melanins; Polyketide Synthases; Photorhabdus; Naphthols; Pigments, Biological
PubMed: 38491909
DOI: 10.1002/advs.202400184 -
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