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Microbial Cell Factories Dec 2022Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer's...
BACKGROUND
Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer's disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear.
RESULTS
The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies on culture plates.
CONCLUSION
The results indicated that the luminescence of luciferase was representative of intracellular NADPH levels. Luciferase can therefore be employed to screen for mutations in genes involved in NADPH regeneration in B. subtilis, and artificial manipulation to enhance NADPH regeneration can promote the production of substances such as scyllo-inositol.
Topics: Glucose 1-Dehydrogenase; NADP; Bacillus subtilis; Luminescence; Inositol; Luciferases
PubMed: 36539761
DOI: 10.1186/s12934-022-01993-0 -
ACS Synthetic Biology Jan 2023Rhabdopeptide/xenortide-like peptide (RXP) nonribosomal peptide synthetases (NRPSs) derived from entomophathogenic and bacteria often produce libraries of different...
Rhabdopeptide/xenortide-like peptide (RXP) nonribosomal peptide synthetases (NRPSs) derived from entomophathogenic and bacteria often produce libraries of different peptides varying in amino acid composition, number and degree of methylation, which mainly is a result of promiscuous docking domains (DDs) mediating protein-protein interactions between the different NRPS subunits. In this study, we present two specific RXP-NRPS systems with rather specific DDs that were used as platforms to generate a series of defined RXPs via the exchange of adenylation/methyltransferase (A-MT) domains in the systems followed by heterologous expression in . Additionally, these results suggest that NRPS subunit interaction is not only exclusively dependent on DDs but at least partially also on A domains.
Topics: Peptides; Peptide Synthases; Amino Acids; Escherichia coli
PubMed: 36535068
DOI: 10.1021/acssynbio.2c00472 -
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 -
BMC Genomics Nov 2022Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with...
BACKGROUND
Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria.
RESULTS
A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified.
CONCLUSIONS
The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.
Topics: Animals; Photorhabdus; Rhabditoidea; Symbiosis; Sequence Analysis, RNA; RNA
PubMed: 36344922
DOI: 10.1186/s12864-022-08952-4 -
Acta Crystallographica. Section F,... Nov 2022Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition...
Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscV) and Aeromonas hydrophila (AscV) are reported. Self-rotation functions revealed that LscV forms oligomers with either eightfold or ninefold symmetry in two different crystal forms. Similarly, AscV was found to exhibit tenfold rotational symmetry. These are the first instances of SctV proteins forming non-nonameric oligomers.
Topics: Type III Secretion Systems; Bacterial Proteins; Crystallography, X-Ray; Molecular Chaperones
PubMed: 36322424
DOI: 10.1107/S2053230X22009736 -
Frontiers in Microbiology 2022The importance of and symbionts to their respective and nematode hosts is that they not only contribute to their entomopathogenicity but also to their fecundity... (Review)
Review
The importance of and symbionts to their respective and nematode hosts is that they not only contribute to their entomopathogenicity but also to their fecundity through the production of small molecules. Thus, this mini-review gives a brief introductory overview of these nematophilic bacteria. Specifically, their type species, nematode hosts, and geographic region of isolations are tabulated. The use of nucleotide sequence-based techniques for their species delineation and how pangenomes can improve this are highlighted. Using the - association as an example, the bacterium-nematode lifecycle is visualized with an emphasis on the role of bacterial biomolecules. Those currently in drug development are discussed, and two potential antimalarial lead compounds are highlighted. Thus, this mini-review tabulates forty-eight significant nematophilic bacteria and visualizes the ecological importance of their biomolecules. It further discusses three of these biomolecules that are currently in drug development. Through it, one is introduced to and bacteria, their natural production of biomolecules in the nematode-bacterium lifecycle, and how these molecules are useful in developing novel therapies.
PubMed: 36187939
DOI: 10.3389/fmicb.2022.993688 -
Nature Oct 2022Entomopathogenic nematodes are widely used as biopesticides. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces...
Entomopathogenic nematodes are widely used as biopesticides. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.
Topics: Animals; Bacterial Toxins; Biological Control Agents; CRISPR-Cas Systems; Culicidae; Drosophila Proteins; Drosophila melanogaster; Fat Body; Gene Editing; Gene Knockdown Techniques; Hemocytes; Humans; Moths; Mucins; Pest Control, Biological; Phagocytosis; Photorhabdus; Repetitive Sequences, Amino Acid; Transgenes; Virulence Factors
PubMed: 36171290
DOI: 10.1038/s41586-022-05250-7 -
Nature Microbiology Oct 2022Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the...
Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its β-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.
Topics: Animals; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Mice; Peptides; Phenylpropionates
PubMed: 36163500
DOI: 10.1038/s41564-022-01227-4 -
PloS One 2022Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial...
Genome analysis of secondary metabolite‑biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.
Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.
Topics: Anti-Bacterial Agents; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Multigene Family; Oxacillin; Photorhabdus; Plant Extracts; Polyketide Synthases; Siderophores; Staphylococcus aureus; Xenorhabdus
PubMed: 36129957
DOI: 10.1371/journal.pone.0274956 -
Nature Chemical Biology Nov 2022The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target...
The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target pathogens and harms the microbiome. This is especially true for Mycobacterium tuberculosis, where treatment requires a 6-month course of antibiotics. Here we show that a novel antimicrobial from Photorhabdus noenieputensis, which we named evybactin, is a potent and selective antibiotic acting against M. tuberculosis. Evybactin targets DNA gyrase and binds to a site overlapping with synthetic thiophene poisons. Given the conserved nature of DNA gyrase, the observed selectivity against M. tuberculosis is puzzling. We found that evybactin is smuggled into the cell by a promiscuous transporter of hydrophilic compounds, BacA. Evybactin is the first, but likely not the only, antimicrobial compound found to employ this unusual mechanism of selectivity.
Topics: Humans; Topoisomerase II Inhibitors; Mycobacterium tuberculosis; DNA Gyrase; Anti-Bacterial Agents; Tuberculosis; Thiophenes; Poisons; Antitubercular Agents
PubMed: 35996001
DOI: 10.1038/s41589-022-01102-7