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Molecular Cell Dec 2021The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active...
The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence-specific unloader MatP. Here, we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE, and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo. Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.
Topics: Adenosine Triphosphatases; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosomes; Chromosomes, Bacterial; Cryoelectron Microscopy; DNA; DNA Repair; DNA-Binding Proteins; Dimerization; Escherichia coli; Escherichia coli Proteins; Genetic Techniques; Genome, Bacterial; Multiprotein Complexes; Photorhabdus; Protein Binding; Protein Conformation; Protein Domains; Repressor Proteins; Cohesins
PubMed: 34739874
DOI: 10.1016/j.molcel.2021.10.011 -
Biology Oct 2021and are considered a global threat to agricultural crops and food security; hence, their control is a critical issue. and nematodes, along with their symbiotic...
and are considered a global threat to agricultural crops and food security; hence, their control is a critical issue. and nematodes, along with their symbiotic bacteria, can achieve the optimal biocontrol agent criterion. Therefore, this study aimed to evaluate the efficacy of , and their symbiotic bacteria ( and ) against and larvae. The virulence of entomopathogenic nematodes (EPNs) was determined at different infective juvenile concentrations and exposure times, while the symbiotic bacteria were applied at the concentration of 3 × 10 colony-forming units (CFU)/mL at different exposure times. Gas chromatography-mass spectrophotometry (GC-MS) analysis and the cytotoxic effect of sp. and sp. were determined. The results indicated that . , . , and their symbiotic bacteria significantly ( ≤ 0.001) induced mortality in both insect species. However, . and its symbiont, sp., were more virulent. Moreover, the data clarified that both symbiotic bacteria outperformed EPNs against but the opposite was true for GC-MS analysis revealed the main active compounds that have insecticidal activity. However, the results revealed that there was no significant cytotoxic effect. In conclusion, . , . , and their symbiotic bacteria can be an optimal option for bio-controlling both insect species. Furthermore, both symbiotic bacteria can be utilized independently on EPNs for the management of both pests, and, hence, they can be safely incorporated into biocontrol programs and tested against other insect pests.
PubMed: 34681098
DOI: 10.3390/biology10100999 -
Chemical Science Sep 2021The development of new antibiotics is imperative to fight increasing mortality rates connected to infections caused by multidrug-resistant (MDR) bacteria. In this...
The development of new antibiotics is imperative to fight increasing mortality rates connected to infections caused by multidrug-resistant (MDR) bacteria. In this context, Gram-negative pathogens listed in the WHO priority list are particularly problematic. Darobactin is a ribosomally produced and post-translationally modified bicyclic heptapeptide antibiotic selectively killing Gram-negative bacteria by targeting the outer membrane protein BamA. The native darobactin A producer HGB1456 shows very limited production under laboratory cultivation conditions. Herein, we present the design and heterologous expression of a synthetically engineered darobactin biosynthetic gene cluster (BGC) in to reach an average darobactin A production titre of 13.4 mg L. Rational design of variants, encoding the darobactin precursor peptide with altered core sequences, resulted in the production of 13 new 'non-natural' darobactin derivatives and 4 previously hypothetical natural darobactins. One of the non-natural compounds, darobactin 9, was more potent than darobactin A, and showed significantly improved activity especially against (0.125 μg mL) and (1-2 μg mL). Importantly, it also displayed superior activity against MDR clinical isolates of . (1-2 μg mL) and (1-4 μg mL). Independent deletions of genes from the darobactin BGC showed that only and , encoding a radical forming -adenosyl-l-methionine-dependent enzyme, are required for darobactin formation. Co-expression of two additional genes associated with the BGCs in hypothetical producer strains identified a proteolytic detoxification mechanism as a potential self-resistance strategy in native producers. Taken together, we describe a versatile heterologous darobactin platform allowing the production of unprecedented active derivatives in good yields, and we provide first experimental evidence for darobactin biosynthesis processes.
PubMed: 34659729
DOI: 10.1039/d1sc02725e -
PeerJ 2021LitR is a master-regulator of transcription in the and quorum sensing (QS) systems of bacteria from and genera. Here, we for the first time directly investigated the...
LitR is a master-regulator of transcription in the and quorum sensing (QS) systems of bacteria from and genera. Here, we for the first time directly investigated the influence of LitR on gene expression in the QS system of psychrophilic bacteria . Investigated promoters were fused with reporter genes cassette in a heterological system of cells, was introduced into the cells under control of P promoter. LitR has been shown to upregulate genes of autoinducer synthase (), luciferase and reductase (), and this effect doesn't depend on presence of gene. To a much lesser degree, LitR induces , but not the the main regulator. Enhanced expression leads to an increase in a LuxI-autoinducer synthesis and a subsequent LuxR-mediated activation of the QS system. Effect of LitR on transcription depends on -box sequence in promoter even in absence of (-box is binding site of LuxR). The last finding indicates a direct interaction of LitR with the promoter in the -box region. Investigation of the effect of LitR on QS systems of mesophilic and psychrophilic showed direct -independent upregulation of and genes. To a lesser degree, it induces and . Therefore, we assume that the main role of LitR in cross-interaction of these three QS systems is stimulating the expression of .
PubMed: 34616599
DOI: 10.7717/peerj.12030 -
Cell Chemical Biology Apr 2022With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to...
With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.
Topics: Bacteria; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Genome, Bacterial; Multigene Family; Recombinases
PubMed: 34508657
DOI: 10.1016/j.chembiol.2021.08.009 -
International Journal of Molecular... Sep 2021Here, we present a new lux-biosensor based on for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have...
Here, we present a new lux-biosensor based on for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the reporter genes are transcribed from the stress-inducible promoters of : the SOS promoter P, the methylation-specific response promoter P, and the oxidative stress promoter P. The luminescence of based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and HO. Comparison with -based lux-biosensors, where the promoters P, P, and P were used, showed generally similar characteristics. However, for P, a higher response amplitude was observed, and for P, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. P and P showed increased sensitivity to the genotoxic effects of the 2,2'-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to -based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.
Topics: Bacillus subtilis; Bacterial Proteins; Biosensing Techniques; Microorganisms, Genetically-Modified; Plasmids; Promoter Regions, Genetic
PubMed: 34502476
DOI: 10.3390/ijms22179571 -
Plants (Basel, Switzerland) Aug 2021The current approaches to sustainable agricultural development aspire to use safer means to control pests and pathogens. bacteria that are insecticidal symbionts of... (Review)
Review
The current approaches to sustainable agricultural development aspire to use safer means to control pests and pathogens. bacteria that are insecticidal symbionts of entomopathogenic nematodes in the genus can provide such a service with a treasure trove of insecticidal compounds and an ability to cope with the insect immune system. This review highlights the need of -derived insecticidal, fungicidal, pharmaceutical, parasiticidal, antimicrobial, and toxic materials to fit into current, or emerging, holistic strategies, mainly for managing plant pests and pathogens. The widespread use of these bacteria, however, has been slow, due to cost, natural presence within the uneven distribution of their nematode partners, and problems with trait stability during in vitro culture. Yet, progress has been made, showing an ability to overcome these obstacles via offering affordable mass production and mastered genome sequencing, while detecting more of their beneficial bacterial species/strains. Their high pathogenicity to a wide range of arthropods, efficiency against diseases, and versatility, suggest future promising industrial products. The many useful properties of these bacteria can facilitate their integration with other pest/disease management tactics for crop protection.
PubMed: 34451705
DOI: 10.3390/plants10081660 -
Life (Basel, Switzerland) Aug 2021Entomopathogenic nematodes (EPNs) are known to be highly pathogenic to insect pests, due to their associated symbiotic bacteria, which produce virulence factors,...
Entomopathogenic nematodes (EPNs) are known to be highly pathogenic to insect pests, due to their associated symbiotic bacteria, which produce virulence factors, exo-enzymes and other harmful secondary metabolites to conquer, kill, and degrade their insect hosts. However, these properties are not fully characterized. This study reports on the antimicrobial activities of sp. strain ETL, symbiotically associated to an insect pathogenic nematode, , against human pathogenic bacteria and toxigenic fungi, as well as the non-targeted profiling of its secondary metabolites (SMs) using gas chromatography coupled to high-resolution time-of-flight mass spectrometry. Fatty acids including 3-eicosene, (E)-; 5-eicosene, (E)-; eicosene; 9-octadecenamide; undecanoic acid with shown antimicrobial activities were detected. This provided more insight on the composition and bioactivities of SMs produced by the sp.
PubMed: 34440531
DOI: 10.3390/life11080787 -
AMB Express Aug 2021Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics,...
Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics, yet several aspects of azasugars remain unknown including their distribution, structural diversity, and chemical ecology. The hallmark signature of bacterial azasugar biosynthesis is a three gene cluster (3GC) coding for aminotransferase, phosphatase, and dehydrogenase enzymes. Using the bioinformatics platform Enzyme Similarity Tool (EST), we identified hundreds of putative three gene clusters coding for azasugar production in microbial species. In the course of this work, we also report a consensus sequence for the aminotransferase involved in azasugar biosynthesis as being: SGNXFRXXXFPNXXXXXXXLXVPXPYCXRC. Most clusters are found in Bacillus and Streptomyces species which typically inhabit soil and the rhizosphere, but some clusters are found with diverse species representation such as Photorhabdus and Xenorhabdus which are symbiotic with entomopathogenic nematodes; the human skin commensal Cutibacterium acnes, and the marine Bacillus rugosus SPB7, a symbiont to the sea sponge Spongia officinalis. This pan-taxonomic survey of the azasugar 3GC signature may lead to the identification of new azasugar producers, facilitate studies of their natural functions, and lead to new potential therapeutics.
PubMed: 34424396
DOI: 10.1186/s13568-021-01279-5 -
PloS One 2021Xenorhabdus and Photorhabdus are gram negative bacteria that can produce several secondary metabolites, including antimicrobial compounds. They have a symbiotic...
Xenorhabdus and Photorhabdus are gram negative bacteria that can produce several secondary metabolites, including antimicrobial compounds. They have a symbiotic association with entomopathogenic nematodes (EPNs). The aim of this study was to isolate and identify Xenorhabdus and Photorhabdus species and their associated nematode symbionts from Northeastern region of Thailand. We also evaluated the antibacterial activity of these symbiotic bacteria. The recovery rate of EPNs was 7.82% (113/1445). A total of 62 Xenorhabdus and 51 Photorhabdus strains were isolated from the EPNs. Based on recA sequencing and phylogeny, Xenorhabdus isolates were identified as X. stockiae (n = 60), X. indica (n = 1) and X. eapokensis (n = 1). Photorhabdus isolates were identified as P. luminescens subsp. akhurstii (n = 29), P. luminescens subsp. hainanensis (n = 18), P. luminescens subsp. laumondii (n = 2), and P. asymbiotica subsp. australis (n = 2). The EPNs based on 28S rDNA and internal transcribed spacer (ITS) analysis were identified as Steinernema surkhetense (n = 35), S. sangi (n = 1), unidentified Steinernema (n = 1), Heterorhabditis indica (n = 39), H. baujardi (n = 1), and Heterorhabditis sp. SGmg3 (n = 3). Antibacterial activity showed that X. stockiae (bMSK7.5_TH) extract inhibited several antibiotic-resistant bacterial strains. To the best of our knowledge, this is the first report on mutualistic association between P. luminescens subsp. laumondii and Heterorhabditis sp. SGmg3. This study could act as a platform for future studies focusing on the discovery of novel antimicrobial compounds from these bacterial isolates.
Topics: Animals; Anti-Bacterial Agents; Drug Resistance, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Larva; Microbial Sensitivity Tests; Nematoda; Photorhabdus; Phylogeny; RNA, Ribosomal, 16S; RNA, Ribosomal, 28S; Soil; Soil Microbiology; Symbiosis; Xenorhabdus
PubMed: 34383819
DOI: 10.1371/journal.pone.0255943