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BioRxiv : the Preprint Server For... Jul 2023Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and...
Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.
PubMed: 37546724
DOI: 10.1101/2023.07.26.550694 -
International Journal of Molecular... Oct 2023The co-culturing of microorganisms is a well-known strategy to study microbial interactions in the laboratory. This approach facilitates the identification of new...
The co-culturing of microorganisms is a well-known strategy to study microbial interactions in the laboratory. This approach facilitates the identification of new signals and molecules produced by one species that affects other species' behavior. In this work, we have studied the effects of the interaction of nine species (, , , , , , , , and ) with the predator bacteria , five of which (, , , , and ) induce mound formation of on complex media (Casitone Yeast extract (CYE) and Casitone tris (CTT); media on which does not form these aggregates under normal culture conditions. An in-depth study on - interactions (the strain producing the strongest effect) has allowed the identification of two siderophores produced by , demethylenenocardamine and nocardamine, responsible for this grouping effect over . Experiments using pure commercial nocardamine and different concentrations of FeSO show that iron depletion is responsible for the behavior of . Additionally, it was found that molecules, smaller than 3 kDa, produced by can induce the production of DK-xanthenes by .
Topics: Myxococcus; Myxococcus xanthus; Streptomyces; Microbial Interactions; Iron
PubMed: 37958645
DOI: 10.3390/ijms242115659 -
Environmental Pollution (Barking, Essex... Aug 2023The potential of sulphur (S), MgSO (Mg), and KHPO (P) in nitrogen retention, ammonia emission decrease, and microbial community succession during composting needs to be...
The potential of sulphur (S), MgSO (Mg), and KHPO (P) in nitrogen retention, ammonia emission decrease, and microbial community succession during composting needs to be investigated. To achieve this, different levels of S (0, 0.2, 0.4, 0.6, and 0.8% in dry weight) plus Mg and P (S + Mg + P) were progressively added in 70 days pig manure aerobic composting. The results revealed that the amendment increased salinity and lowered pH and dephytotoxication of the product with the increase of S amount. However, no significant inhibition effects were observed on the evolution of the thermophilic phase and product maturity. In addition, the amendment significantly reduced the total NH and NO emissions by 29.66%-58.81% and 20.6%-56.7%, increased NH level by 17.22%-73.21% in thermophilic phase and NO content by 26.17%-57.48% in a mature phase, and elevated the total Kjeldahl nitrogen content by 34.28%-46.6% during the composting. In addition, compared to the control, the supplement markedly encouraged the formation of guanite in the compost product. The S addition stimulated the growth of Anseongella, Actinomadura, Chelativorans, Castellaniella, Luteimonas, and Steroidobacter microbial communities which functioned well in the degradation of nitrogen-containing compounds and organic matter. Evidence from Redundancy Analysis, Firmicutes, Myxococcus, Chloroflexi, Gemmatimonadota, and Deinococcota showed positive correlations with pH. These results imply that adding S-Mg-P amendment encourages the population and activity of specific functional microorganisms, and facilitated the ammonia emission reduction by lowering pH and thus reserved nitrogen through the formation of guanite during composting. The investigation of bacterial community abundance and environmental variables at the phylum and genus levels over time revealed that adding of 0.6% S in conjunction with P and Mg minerals was suitable for nitrogen loss mitigation in composting. The findings suggest using S + Mg + P supplement to conserve nitrogen in pig dung aerobic composting.
Topics: Swine; Animals; Ammonia; Manure; Composting; Soil; Nitrogen; Sulfur; Bacteria
PubMed: 37263560
DOI: 10.1016/j.envpol.2023.121934 -
MSystems Jun 2024The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and...
UNLABELLED
The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery.
IMPORTANCE
Serving as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications.
Topics: CRISPR-Cas Systems; Genome, Bacterial; Myxococcales; Phylogeny; Clustered Regularly Interspaced Short Palindromic Repeats
PubMed: 38747603
DOI: 10.1128/msystems.01210-23 -
Nature Communications Sep 2023Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that...
Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), collapses rod shape despite aPBPs being non-essential for rod-like morphology in the bacterium Myxococcus xanthus. Here, we demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase. Moenomycin promotes the binding between DacB and PG and thus reduces the mobility of DacB through PBP1a2. Conversely, DacB also regulates the distribution and dynamics of aPBPs. Our findings clarify the action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.
Topics: Peptidoglycan; Bambermycins; Nitric Oxide Synthase; Peptide Hydrolases; Cell Wall; Myxococcus xanthus; Penicillin-Binding Proteins
PubMed: 37660104
DOI: 10.1038/s41467-023-41082-3 -
RSC Chemical Biology Nov 20238-Azido-3,8-dideoxy-α/β-d--oct-2-ulosonic acid (Kdo-8-N) is a Kdo derivative used in metabolic labeling of lipopolysaccharide (LPS) structures found on the cell...
8-Azido-3,8-dideoxy-α/β-d--oct-2-ulosonic acid (Kdo-8-N) is a Kdo derivative used in metabolic labeling of lipopolysaccharide (LPS) structures found on the cell membrane of Gram-negative bacteria. Several studies have reported successful labeling of LPS using Kdo-8-N and visualization of LPS by a fluorescent reagent through click chemistry on a selection of Gram-negative bacteria such as strains, , and . Motivated by the promise of Kdo-8-N to be useful in the investigation of LPS biosynthesis and cell surface labeling across different strains, we set out to explore the variability in nature and efficiency of LPS labeling using Kdo-8-N in a variety of strains and serotypes. We optimized the chemical synthesis of Kdo-8-N and subsequently used Kdo-8-N to metabolically label pathogenic strains from commercial and clinical origin. Interestingly, different extents of labeling were observed in different strains, which seemed to be dependent also on growth media, and the majority of labeled LPS appears to be of the 'rough' LPS variant, as visualized using SDS-PAGE and fluorescence microscopy. This knowledge is important for future application of Kdo-8-N in the study of LPS biosynthesis and dynamics, especially when working with clinical isolates.
PubMed: 37920390
DOI: 10.1039/d3cb00110e -
Proceedings of the National Academy of... May 2024Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. encapsulins protect the bacterial cells against oxidative...
Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe to Fe for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no β-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.
Topics: Myxococcus xanthus; Bacterial Proteins; FMN Reductase; Crystallography, X-Ray; Flavin Mononucleotide; Iron; Models, Molecular; Cryoelectron Microscopy
PubMed: 38748579
DOI: 10.1073/pnas.2400426121 -
Experimental Biology and Medicine... 2024Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of...
Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.
Topics: Mesenchymal Stem Cells; Transferrin; Humans; Myxococcus xanthus; Endocytosis; Receptors, Transferrin; Luminescent Proteins
PubMed: 38774281
DOI: 10.3389/ebm.2024.10055 -
Bio-protocol Jul 2023In the environment, bacteria compete for niche occupancy and resources; they have, therefore, evolved a broad variety of antibacterial weapons to destroy competitors....
In the environment, bacteria compete for niche occupancy and resources; they have, therefore, evolved a broad variety of antibacterial weapons to destroy competitors. Current laboratory techniques to evaluate antibacterial activity are usually labor intensive, low throughput, costly, and time consuming. Typical assays rely on the outgrowth of colonies of prey cells on selective solid media after competition. Here, we present fast, inexpensive, and complementary optimized protocols to qualitatively and quantitively measure antibacterial activity. The first method is based on the degradation of a cell-impermeable chromogenic substrate of the β-galactosidase, a cytoplasmic enzyme released during lysis of the attacked reporter strain. The second method relies on the lag time required for the attacked cells to reach a defined optical density after the competition, which is directly dependent on the initial number of surviving cells. Key features First method utilizes the release of β-galactosidase as a proxy for bacterial lysis. Second method is based on the growth timing of surviving cells. Combination of two methods discriminates between cell death and lysis, cell death without lysis, or survival to quasi-lysis. Methods optimized to various bacterial species such as , and . Graphical overview.
PubMed: 37449039
DOI: 10.21769/BioProtoc.4706 -
Frontiers in Microbiology 2024This study aimed to study the characterization and the potential lipid-lowering effects of new isolated lactic acid bacteria from the feces of healthy adult cats.
INTRODUCTION
This study aimed to study the characterization and the potential lipid-lowering effects of new isolated lactic acid bacteria from the feces of healthy adult cats.
METHODS
We collected 85 cat fecal samples, isolated, screening lactic acid bacteria strains from samples, and investigated their and biological properties.
RESULTS
A total of 221 lactic acid bacteria strains were isolated from 85 cat fecal samples. Sixteen strains with calcium dissolution rings greater than 1 mm were identified and selected for further characterization. Three lactic acid bacteria strains, L-27-2, L-14-1, and , were identified as showing the most promising rates of cholesterol degradation (greater than 20%) and bacteriostatic radius (over 15 mm). These three strains exhibited robust growth and adherence to epithelial cells, along with adaptability to low pH (greater than 70%) and high bile salt conditions (greater than 60%), and remarkable cholesterol degradation and anti-pathogen activity. Sixteen mice were fed a high-fat diet (HFD) from 4 to 8 weeks of age, while a control group of the same size received a normal diet (ND). At 8 weeks of age, serum, feces and adipose tissue were collected. The results showed that, compared with mice fed an HFD diet alone, all mice fed an HFD diet plus lactic acid bacteria could decrease weight gain. < 0.05 and the pathological changes of adipose tissue were alleviated. In addition, mice fed L-14-1 and F203 showed abdominal fat accumulation decreased ( < 0.05). Mice fed L-27-2 showed serum and liver triglyceride (TG) decreased ( < 0.05) and mice fed F203 showed serum high density lipoprotein cholesterol (HDL-C) increased ( < 0.01). mice fed L-27-2 and L-14-1 showed inflammatory cytokines (IL-6) was decreased ( < 0.01) Analysis of the fecal microbiota of mice fed these three lactic acid bacteria strains revealed alterations in the gut microbial community. There were common changes in intestinal microbes in mice fed these three lactic acid bacteria: (1) decreased; (2) increased; (3) decreased. The microbes mentioned are all part of the core intestinal flora.
DISCUSSION
This study provided three potential lactic acid bacteria for alleviating animal obesity and inflammation.
PubMed: 38721604
DOI: 10.3389/fmicb.2024.1392864