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Cell Chemical Biology Aug 2023Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target...
Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit in vivo activity against drug-resistant pathogens. First isolated from Photorhabdus species, the corresponding biosynthetic gene clusters (BGCs) are widespread among γ-proteobacteria, including the genera Vibrio, Yersinia, and Pseudoalteromonas (P.). While the organization of the BGC core is highly conserved, a small subset of Pseudoalteromonas carries an extended BGC with additional genes. Here, we report the identification of brominated and dehydrated darobactin derivatives from P. luteoviolacea strains. The marine derivatives are active against multidrug-resistant (MDR) Gram-negative bacteria and showed solubility and plasma protein binding ability different from darobactin A, rendering it more active than darobactin A. The halogenation reaction is catalyzed by DarH, a new class of flavin-dependent halogenases with a novel fold.
Topics: Phenylpropionates; Gram-Negative Bacteria; Metabolome
PubMed: 37451267
DOI: 10.1016/j.chembiol.2023.06.011 -
Bio-protocol Jul 2023The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs)...
The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.
PubMed: 37449040
DOI: 10.21769/BioProtoc.4709 -
Plant Disease Nov 2023Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our...
Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our research team showed that the combination of two nematicidal secondary metabolites (SMs) derived from bacteria, -cinnamic acid (-CA), and (4)-5-phenylpent-4-enoic acid (PPA) have a synergistic effect against RKNs in vitro. In this study, we considered in planta assays to assess the effects of this SM mixture on the virulence and reproductive fitness of the RKN in a cowpea. Factorial combinations of five -CA + PPA concentrations (0, 9.0, 22.9, 57.8, and 91.0 μg/ml) and two nematode inoculation conditions (presence or absence) were evaluated in 6-week growth chamber experiments. Results from this study showed that a single root application of the -CA + PPA mixture significantly reduced the penetration of infective juveniles (J2s) into the cowpea roots. The potential toxicity of -CA + PPA on RKN-susceptible cowpea seedlings was also investigated. The effect of -CA + PPA × nematode inoculation interactions and the -CA + PPA mixture did not show significant phytotoxic effects, nor did it adversely affect plant growth parameters or alter leaf chlorophyll content. Total leaf chlorophyll and chlorophyll content were significantly reduced (by 15 and 22%, respectively) only by the nematode inoculum and not by any of the SM treatments. Our results suggest that a single root application of a mixture of -CA and PPA reduces J2's ability to infect the roots without impairing plant growth or chlorophyll content.
Topics: Animals; Tylenchoidea; Vigna; Photorhabdus; Antinematodal Agents; Chlorophyll
PubMed: 37330631
DOI: 10.1094/PDIS-11-22-2574-SC -
Heliyon Jun 2023The Rcs sensor system, comprising the RcsB/RcsC/RcsD and RcsF proteins, is used by bacteria of the order to withstand envelope damage. In non-stress conditions, Rcs is...
The Rcs sensor system, comprising the RcsB/RcsC/RcsD and RcsF proteins, is used by bacteria of the order to withstand envelope damage. In non-stress conditions, Rcs is repressed by IgaA, a membrane protein with three cytoplasmic regions (cyt-1, cyt-2 and cyt-3). How the Rcs-IgaA axis evolved within has not been yet explored. Here, we report phylogenetic data supporting co-evolution of IgaA with RcsC/RcsD. Functional exchange assays showed that IgaA from and , but not from or the endosymbionts and , repress the Rcs system of . IgaA from , however, repress only partially the Rcs system despite being produced at high levels in the complementation assay. The modelled structures of these IgaA variants uncovered one periplasmic and two cytoplasmic conserved β-rich architectures forming partially closed small β-barrel (SBB) domains. Conserved residues map in a connector linking cytoplasmic SSB-1 and SBB-2 domains (E180-R265); a region of cyt-1 facing cyt-2 (R188-E194-D309 and T191-H326); and between cyt-2 and cyt-3 (H293-E328-R686). These structures validated early studies in that assigned a role in function to R188, T191 and G262, and in addition revealed a previously unnoticed "hybrid" SBB-2 domain to which cyt-1 and cyt-2 contribute. IgaA variants not functional or partially functional in lack H192-P249 and R255-D313 interactions. Among these variants, only IgaA from conserves the helix α6 in SSB-1 that is present in IgaA from and . RcsF and RcsD, which interact directly with IgaA, failed to show structural features linked to specific IgaA variants. Altogether, our data provide new insights into IgaA by mapping residues selected differently during evolution and involved in function. Our data also infer contrasting lifestyles of bacteria as source of variability in the IgaA-RcsD/IgaA-RcsF interactions.
PubMed: 37303533
DOI: 10.1016/j.heliyon.2023.e16661 -
Journal of Agricultural and Food... Jun 2023Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against . However, the commercial development of Xcn1 is hindered by the low yield, which results in...
Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against . However, the commercial development of Xcn1 is hindered by the low yield, which results in high application costs. In this study, multiple metabolic strategies, including blocking the degradation pathway, promoter engineering, and deletion of competing biosynthetic gene clusters, were employed to improve the production of Xcn1, which was increased from 0.07 to 0.91 g/L. The formation of Xcn1 reached 1.94 g/L in the TB medium with the final strain T3 in a shake flask and further reached 3.52 g/L in a 5 L bioreactor, which is the highest yield ever reported. The engineered strain provides a valuable platform for production of Xcn1, and the possible commercial development of the biofungicide. We anticipate that the metabolic engineering strategies utilized in this study and the constructed constitutive promoter library can be widely applied to other bacteria of the genera and .
Topics: Xenorhabdus; Anti-Infective Agents; Benzopyrans; Bioreactors
PubMed: 37278378
DOI: 10.1021/acs.jafc.3c01793 -
Heliyon May 2023Significant progress has been made in recent years on exploring immunometabolism, a field that integrates two processes essential for maintaining tissue and organismal...
Significant progress has been made in recent years on exploring immunometabolism, a field that integrates two processes essential for maintaining tissue and organismal homeostasis, immunity and metabolism. The nematode parasite , its mutualistic bacteria , and the fruit fly constitute a unique system to investigate the molecular basis of host immunometabolic response to nematode-bacterial complexes. In this study, we explored the contribution of the two major immune signaling pathways, Toll and Imd, to sugar metabolism in larvae during infection with nematodes. We infected Toll or Imd signaling loss-of-function mutant larvae with nematodes and assessed larval survival ability, feeding rate, and sugar metabolism. We found no significant differences in the survival ability or levels of sugar metabolites in any of the mutant larvae when responding to infection. However, we found that the Imd mutant larvae have higher feeding rate than controls during the early stages of infection. In addition, feeding rates are lower in Imd mutants relative to the control larvae as the infection progresses. We further showed that and gene expression increases in Imd mutants compared to controls early in the infection, but their expression levels decrease at later times. These findings indicate that Imd signaling activity regulates the feeding rate and and expression in larvae infected with . Results from this study facilitate our understanding of the link between host innate immunity and sugar metabolism in the context of infectious diseases caused by parasitic nematodes.
PubMed: 37251825
DOI: 10.1016/j.heliyon.2023.e16139 -
Acta Crystallographica. Section D,... Jun 2023Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected...
Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX-SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.
Topics: Protein Binding; Molecular Chaperones; Bacterial Proteins; Yersinia; Hydrophobic and Hydrophilic Interactions
PubMed: 37204817
DOI: 10.1107/S2059798323003248 -
Research in Microbiology 2023The Resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump contributes to multidrug resistance in Gram-negative bacteria. Recently, the bacterium Photorhabdus...
The Resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump contributes to multidrug resistance in Gram-negative bacteria. Recently, the bacterium Photorhabdus laumondii TT01 has emerged as a goldmine for novel anti-infective drug discovery. Outside plants, Photorhabdus is the only Gram-negative known to produce stilbene-derivatives including 3,5-dihydroxy-4-ethyl-trans-stilbene and 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS). IPS is a bioactive polyketide which received considerable attention, mainly because of its antimicrobial properties, and is currently in late-stage clinical development as a topical treatment for psoriasis and dermatitis. To date, little is known about how Photorhabdus survives in the presence of stilbenes. We combined genetic and biochemical approaches to assess whether AcrAB efflux pump exports stilbenes in P. laumondii. We demonstrated that the wild-type (WT) exerts an antagonistic activity against its derivative ΔacrA mutant, and that is able to outcompete it in a dual-strain co-culture assay. The ΔacrA mutant also showed high sensitivity to 3,5-dihydroxy-4-ethyl-trans-stilbene and IPS as well as decreased IPS concentrations in its supernatant comparing to the WT. We report here a mechanism of self-resistance against stilbene derivatives of P. laumondii TT01, which enables these bacteria to survive under high concentrations of stilbenes by extruding them out via the AcrAB efflux pump.
PubMed: 37196776
DOI: 10.1016/j.resmic.2023.104081 -
International Journal of Systematic and... May 2023Six Gram-negative, rod-shaped bacterial strains isolated from entomopathogenic nematodes were characterized to determine their taxonomic position. 16S rRNA and gene...
Six Gram-negative, rod-shaped bacterial strains isolated from entomopathogenic nematodes were characterized to determine their taxonomic position. 16S rRNA and gene sequences indicate that they belong to the class , family and genus , and that some of them are conspecifics. Two of them, APURE and JAR, were selected for further molecular characterization using whole genome- and whole-proteome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions using whole genome and whole proteome sequences show that strains APURE and JAR are closely related to subsp. ATCC 29999 and to subsp. MEX47-22. Moreover, digital DNA-DNA hybridization (dDDH) values between APURE and subsp. ATCC 29999, APURE and subsp. MEX47-22, and APURE and JAR are 61.6, 61.2 and 64.1 %, respectively. These values are below the 70 % divergence threshold that delimits prokaryotic species. dDDH scores between JAR and subsp. ATCC 29999 and between JAR and subsp. MEX47-22 are 71.9 and 74.8 %, respectively. These values are within the 70 and 79 % divergence thresholds that delimit prokaryotic subspecies. Based on these genomic divergence values, APURE and JAR represent two different taxa, for which we propose the names: sp. nov. with APURE (=CCM 9236 =CCOS 2019) as type strain and subsp. subsp. nov. with JAR (=CCM 9235 =CCOS 2021) as type strain. Our study contributes to a better understanding of the biodiversity of an important bacterial group with enormous biotechnological and agricultural potential.
Topics: Animals; Photorhabdus; Phylogeny; RNA, Ribosomal, 16S; Proteome; Bacterial Typing Techniques; Sequence Analysis, DNA; DNA, Bacterial; Base Composition; Fatty Acids; Nematoda
PubMed: 37171451
DOI: 10.1099/ijsem.0.005872 -
PLoS Pathogens May 2023Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed... (Review)
Review
Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.
Topics: Animals; Photorhabdus; Penaeidae; Bacterial Proteins; Bacterial Toxins; Insecta; Vibrio parahaemolyticus
PubMed: 37141203
DOI: 10.1371/journal.ppat.1011330