-
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 -
Molecular Biology and Evolution May 2024Prokaryotes dominate the Tree of Life, but our understanding of the macroevolutionary processes generating this diversity is still limited. Habitat transitions are...
Prokaryotes dominate the Tree of Life, but our understanding of the macroevolutionary processes generating this diversity is still limited. Habitat transitions are thought to be a key driver of prokaryote diversity. However, relatively little is known about how prokaryotes successfully transition and persist across environments, and how these processes might vary between biomes and lineages. Here, we investigate biome transitions and specialization in natural populations of a focal bacterial phylum, the Myxococcota, sampled across a range of replicated soils and freshwater and marine sediments in Cornwall (UK). By targeted deep sequencing of the protein-coding gene rpoB, we found >2,000 unique Myxococcota lineages, with the majority (77%) classified as biome specialists and with only <5% of lineages distributed across the salt barrier. Discrete character evolution models revealed that specialists in one biome rarely transitioned into specialists in another biome. Instead, evolved generalism mediated transitions between biome specialists. State-dependent diversification models found variation in speciation rates across the tree, but this variation was independent of biome association or specialization. Our findings were robust to phylogenetic uncertainty, different levels of species delineation, and different assumed amounts of unsampled diversity resulting in an incomplete phylogeny. Overall, our results are consistent with a "jack-of-all-trades" tradeoff where generalists suffer a cost in any individual environment, resulting in rapid evolution of niche specialists and shed light on how bacteria could transition between biomes.
Topics: Myxococcales; Biological Evolution; Ecosystem; Phylogeny; Genetic Speciation
PubMed: 38717941
DOI: 10.1093/molbev/msae088 -
Proceedings of the National Academy of... Apr 2024Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are...
Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the T4aP (T4aP) at a resolution of 3.0 Å using cryo-EM. The T4aP follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aP is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aP variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.
Topics: Fimbriae Proteins; Myxococcus xanthus; Fimbriae, Bacterial; Protein Structure, Secondary; Virulence
PubMed: 38625941
DOI: 10.1073/pnas.2321989121 -
Protein Science : a Publication of the... May 2024Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV...
Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the β-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.
Topics: Myxococcus xanthus; Adenosine Triphosphatases; Bacterial Proteins; Translesion DNA Synthesis; Escherichia coli; DNA; DNA Replication
PubMed: 38591662
DOI: 10.1002/pro.4981 -
The responses of CO emission to nitrogen application and earthworm addition in the soybean cropland.PeerJ 2024The effects of nitrogen application or earthworms on soil respiration in the Huang-Huai-Hai Plain of China have received increasing attention. However, the response of...
The effects of nitrogen application or earthworms on soil respiration in the Huang-Huai-Hai Plain of China have received increasing attention. However, the response of soil carbon dioxide (CO) emission to nitrogen application and earthworm addition is still unclear. A field experiment with nitrogen application frequency and earthworm addition was conducted in the Huang-Huai-Hai Plain. Results showed nitrogen application frequency had a significant effect on soil respiration, but neither earthworms nor their interaction with nitrogen application frequency were significant. Low-frequency nitrogen application (NL) significantly increased soil respiration by 25%, while high-frequency nitrogen application (NH), earthworm addition (E), earthworm and high-frequency nitrogen application (E*NH), and earthworm and low-frequency nitrogen application (E*NL) also increased soil respiration by 21%, 21%, 12%, and 11%, respectively. The main reason for the rise in soil respiration was alterations in the bacterial richness and keystone taxa (Myxococcales). The NH resulted in higher soil nitrogen levels compared to NL, but NL had the highest bacterial richness. The abundance of Corynebacteriales and Gammaproteobacteria were positively connected with the CO emissions, while Myxococcales, Thermoleophilia, and Verrucomicrobia were negatively correlated. Our findings indicate the ecological importance of bacterial communities in regulating the carbon cycle in the Huang-Huai-Hai Plain.
Topics: Animals; Oligochaeta; Carbon Dioxide; Glycine max; Nitrogen; Soil; Myxococcales; Crops, Agricultural
PubMed: 38560479
DOI: 10.7717/peerj.17176 -
Genome Biology and Evolution May 2024Intrinsic rates of genetic mutation have diverged greatly across taxa and exhibit statistical associations with several other parameters and features. These include...
Intrinsic rates of genetic mutation have diverged greatly across taxa and exhibit statistical associations with several other parameters and features. These include effective population size (Ne), genome size, and gametic multicellularity, with the latter being associated with both increased mutation rates and decreased effective population sizes. However, data sufficient to test for possible relationships between microbial multicellularity and mutation rate (µ) are lacking. Here, we report estimates of two key population-genetic parameters, Ne and µ, for Myxococcus xanthus, a bacterial model organism for the study of aggregative multicellular development, predation, and social swarming. To estimate µ, we conducted an ∼400-day mutation accumulation experiment with 46 lineages subjected to regular single colony bottlenecks prior to clonal regrowth. Upon conclusion, we sequenced one clonal-isolate genome per lineage. Given collective evolution for 85,323 generations across all lines, we calculate a per base-pair mutation rate of ∼5.5 × 10-10 per site per generation, one of the highest mutation rates among free-living eubacteria. Given our estimate of µ, we derived Ne at ∼107 from neutral diversity at four-fold degenerate sites across two dozen M. xanthus natural isolates. This estimate is below average for eubacteria and strengthens an already clear negative correlation between µ and Ne in prokaryotes. The higher and lower than average mutation rate and Ne for M. xanthus, respectively, amplify the question of whether any features of its multicellular life cycle-such as group-size reduction during fruiting-body development-or its highly structured spatial distribution have significantly influenced how these parameters have evolved.
Topics: Myxococcus xanthus; Mutation Rate; Population Density; Genome, Bacterial
PubMed: 38526062
DOI: 10.1093/gbe/evae066 -
The Journal of Biological Chemistry Apr 2024Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one...
Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB or MglB linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB, but not MglB, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB and β of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.
Topics: Bacterial Proteins; Enzyme Activation; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Myxococcus xanthus; Protein Multimerization; Models, Molecular; Protein Structure, Quaternary
PubMed: 38508314
DOI: 10.1016/j.jbc.2024.107197 -
The Science of the Total Environment Apr 2024This study aimed to assess the potential impact of long-term chronic exposure (69 years) to naturally-occurring radionuclides (RNs) and heavy metals on microbial...
This study aimed to assess the potential impact of long-term chronic exposure (69 years) to naturally-occurring radionuclides (RNs) and heavy metals on microbial communities in sediment from a stream flowing through a watershed impacted by an ancient mining site (Rophin, France). Four sediment samples were collected along a radioactivity gradient (for U368 to 1710 Bq.Kg) characterized for the presence of the bioavailable fractions of radionuclides (Ra, Po), and trace metal elements (Th, U, As, Pb, Cu, Zn, Fe). Results revealed that the available fraction of contaminants was significant although it varied considerably from one element to another (0 % for As and Th, 5-59 % for U). Nonetheless, microbial communities appeared significantly affected by such chronic exposure to (radio)toxicities. Several microbial functions carried by bacteria and related with carbon and nitrogen cycling have been impaired. The high values of fungal diversity and richness observed with increasing downstream contamination (H' = 4.4 and Chao1 = 863) suggest that the community had likely shifted toward a more adapted/tolerant one as evidenced, for example, by the presence of the species Thelephora sp. and Tomentella sp. The bacterial composition was also affected by the contaminants with enrichment in Myxococcales, Acidovorax or Nostocales at the most contaminated points. Changes in microbial composition and functional structure were directly related to radionuclide and heavy metal contaminations, but also to organic matter which also significantly affected, directly or indirectly, bacterial and fungal compositions. Although it was not possible to distinguish the specific effects of RNs from heavy metals on microbial communities, it is essential to continue studies considering the available fraction of elements, which is the only one able to interact with microorganisms.
Topics: Metals, Heavy; Microbiota; Bacteria; Radioisotopes; France
PubMed: 38325491
DOI: 10.1016/j.scitotenv.2024.170692 -
Microbiology Spectrum Mar 2024In the past century, microbial natural products have proven themselves to be substantial and fruitful sources of anti-infectives. In addition to the well-studied...
In the past century, microbial natural products have proven themselves to be substantial and fruitful sources of anti-infectives. In addition to the well-studied Actinobacteria, understudied bacterial taxa like the Gram-negative myxobacteria have increasingly gained attention in the ongoing search for novel and biologically active natural products. In the course of a regional sampling campaign to source novel myxobacteria, we recently uncovered new myxobacterial strains MCy12716 and MCy12733 belonging to the clade. Early bioactivity screens of the bacterial extracts revealed the presence of bioactive natural products that were identified as angiolam A and several novel derivatives. Sequencing of the corresponding producer strains allowed the identification of the angiolam biosynthetic gene cluster, which was verified by targeted gene inactivation. Based on bioinformatic analysis of the biosynthetic gene cluster, a concise biosynthesis model was devised to explain angiolam biosynthesis. Importantly, novel angiolam derivatives uncovered in this study named angiolams B, C, and D were found to display promising antiparasitic activities against the malaria pathogen in the 0.3-0.8 µM range.IMPORTANCEThe COVID-19 pandemic and continuously emerging antimicrobial resistance (AMR) have recently raised awareness about limited treatment options against infectious diseases. However, the shortage of treatment options against protozoal parasitic infections, like malaria, is much more severe, especially for the treatment of so-called neglected tropical diseases. The detection of anti-parasitic bioactivities of angiolams produced by MCy12716 and MCy12733 displays the hidden potential of scarcely studied natural products to have promising biological activities in understudied indications. Furthermore, the improved biological activities of novel angiolam derivatives against and the evaluation of its biosynthesis display the opportunities of the angiolam scaffold on route to treat protozoal parasitic infections as well as possible ways to increase the production of derivatives with improved bioactivities.
Topics: Humans; Myxococcales; Antiparasitic Agents; Pandemics; Malaria, Falciparum; Plasmodium falciparum; Biological Products
PubMed: 38298128
DOI: 10.1128/spectrum.03689-23 -
PLoS Biology Jan 2024Ecological variation influences the character of many biotic interactions, but examples of predator-prey reversal mediated by abiotic context are few. We show that the...
Ecological variation influences the character of many biotic interactions, but examples of predator-prey reversal mediated by abiotic context are few. We show that the temperature at which prey grow before interacting with a bacterial predator can determine the very direction of predation, reversing predator and prey identities. While Pseudomonas fluorescens reared at 32°C was extensively killed by the generalist predator Myxococcus xanthus, P. fluorescens reared at 22°C became the predator, slaughtering M. xanthus to extinction and growing on its remains. Beyond M. xanthus, diffusible molecules in P. fluorescens supernatant also killed 2 other phylogenetically distant species among several examined. Our results suggest that the sign of lethal microbial antagonisms may often change across abiotic gradients in natural microbial communities, with important ecological and evolutionary implications. They also suggest that a larger proportion of microbial warfare results in predation-the killing and consumption of organisms-than is generally recognized.
Topics: Animals; Predatory Behavior; Antibiosis; Biological Evolution; Microbiota; Myxococcus xanthus
PubMed: 38261596
DOI: 10.1371/journal.pbio.3002454