Manipulating subcellular protein localization to enhance target protein accumulation in minicells

Journal of Biological Engineering, Journal Year: 2025, Volume and Issue: 19(1)

Published: March 29, 2025

Minicells are chromosome-free derivatives of bacteria formed through irregular cell division. Unlike simplified structures, minicells retain all cellular components the parent except for chromosome. This feature reduces immunogenic responses, making them advantageous various biotechnological applications, including chemical production and drug delivery. To effectively utilize minicells, it is essential to ensure accumulation target proteins within them, enhancing their efficiency as delivery vehicles. In this study, we engineered Escherichia coli by deleting minCD genes, generating minicell-producing strains, investigated strategies enhance protein minicells. Comparative proteomic analysis revealed that most parent-cell but exhibit an asymmetric proteome distribution, leading selective enrichment. We demonstrated heterologous proteins, such GFP RFP, accumulate more abundantly in than cells, regardless expression levels. further accumulation, manipulated localization fusing polar signals. While fused with PtsI Tsr exhibited 2.6-fold 2.8-fold increases respectively, fusion PopZ resulted a remarkable 15-fold increase concentration under low induction conditions. These findings highlight critical role spatial organization cargo-loading capabilities By leveraging signals, work provides robust framework optimizing efficient carriers diverse from therapeutic industrial biomanufacturing.

Language: Английский

---

Morphological and Biochemical Characterization of a Cellulose-Degrading Bacteria isolated from Horse Feces (Equus caballus)

IOP Conference Series Earth and Environmental Science, Journal Year: 2025, Volume and Issue: 1484(1), P. 012036 - 012036

Published: April 1, 2025

Abstract Cellulase is a widely developed enzyme for industrial applications, produced by various sources, including bacteria. While numerous bacteria are known to produce cellulose-degrading enzymes (CDE), further exploration needed discover unique and highly active CDEs. This study focuses on screening, identifying, characterizing (CDB) from horse faces, which considered an excellent source such Fecal samples were collected Megastar Horse Farm in Batu, Malang, screened carboxymethyl cellulose (CMC)-containing agar, incubated at 37 °C. The colonies that formed halo zones the agar isolated, their genomic DNA was extracted 16S rRNA gene amplification. Sequence analysis revealed isolate showed over 98% similarity B. amyloliquefaciens SLBD subsequently labeled as SLBD. Gram staining scanning electron microscopy confirmed Gram-positive with rod-shaped morphology. also grew optimally Luria-Bertani (LB) medium CDE activity of this strain assessed using its extracellular fraction against CMC mannan oligosaccharides (MOS), revealing substrate-dependent profile, being preferred substrate.

Language: Английский

---

Cell-free protein synthesis from non-growing, stressed Escherichia coli

Scientific Reports, Journal Year: 2017, Volume and Issue: 7(1)

Published: Nov. 22, 2017

Cell-free protein synthesis is a versatile production system. Performance of the depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they routinely obtained exponential growing cells, aiming capture most translation Here, we report an cell-free system derived cells harvested at non-growth, stressed conditions. We found downshift ribosomes and proteins. However, characterization revealed that stoichiometry key factors was conserved, pointing fully intact This emphasized by rates, which were comparable those systems fast-growing cells. Our approach less laborious than traditional extract preparation methods multiplies yield per cultivation. simplified growth protocol has potential attract new entrants broaden pool applications. In this respect, originating heat stressed, non-growing enabled extension endogenous transcription units. demonstrated sigma factor depending activation parallel transcription. expression platform adds existing versatility presents tool for biology.

Language: Английский

---

Growth and Extended Survival of Escherichia coli O157:H7 in Soil Organic Matter

Frontiers in Microbiology, Journal Year: 2018, Volume and Issue: 9

Published: April 23, 2018

Enterohaemorrhagic Escherichia coli, such as serotype O157:H7, are a leading cause of food-associated outbreaks. While the primary reservoir is associated with cattle, plant foods have been sources human infection. E. coli able to grow in tissue food plants spinach. fecal contamination suspect, soil has underestimated potential reservoir. Persistence bacterial populations open systems product growth, death, predation, and competition. Here we report that O157:H7 can using soluble compounds soil, characterize effect growth stationary phase proteome. 933D (stxII-) was cultured Soil Extracted Soluble Organic Matter (SESOM) culturable count determined for 24 d. The proteomes exponential were characterized by 2D gel electrophoresis protein spots identified MALDI-TOF mass spectrometry. LB controls displayed death phase, SESOM grown population remained d, indicating an altered physiological state superior longevity. This not due decreased cell density on entry 24h concentrated 10-fold retained their Principal component analysis showed from different. Differences included proteins involved stress response, motility, membrane wall composition, nutrient uptake, translation turnover, anabolic catabolic pathways, soil-grown cells entering phase. results suggest may be commensal that, absence predation competition, maintains stable soil.

Language: Английский

---

Awakening the natural capability of psicose production in Escherichia coli

npj Science of Food, Journal Year: 2023, Volume and Issue: 7(1)

Published: Oct. 14, 2023

Due to the rampant rise in obesity and diabetes, consumers are desperately seeking for ways reduce their sugar intake, but date there no options that both accessible without sacrifice of palatability. One most promising new ingredients food system as a non-nutritive substitute with near perfect palatability is D-psicose. D-psicose currently produced using an vitro enzymatic isomerization D-fructose, resulting low yield purity, therefore requiring substantial downstream processing obtain high purity product. This has made adoption into products limited results significantly higher per unit costs, reducing accessibility those need. Here, we found Escherichia coli natively possesses thermodynamically favorable pathway produce from D-glucose through series phosphorylation-epimerization-dephosphorylation steps. To increase carbon flux towards production, introduced genetic modifications enzymes, central metabolism, competing metabolic pathways. In attempt maximize cellular viability implemented methods dynamic regulation key genes including clustered regularly interspaced short palindromic repeats inhibition (CRISPRi) stationary-phase promoters. The engineered strains achieved complete consumption production D-psicose, at titer 15.3 g L-1, productivity 2 L-1 h-1, 62% under test tube conditions. These demonstrate whole-cell catalysis sustainable alternative synthesis

Language: Английский

---

Controlled burn: interconnections between energy-spilling pathways and metabolic signaling in bacteria

Journal of Bacteriology, Journal Year: 2025, Volume and Issue: unknown

Published: March 31, 2025

ABSTRACT Bacterial energy-spilling pathways—such as overflow metabolism and futile cycles—have been considered inefficient forms of that result from poor regulatory control or function mechanisms to cope with excess energy. However, mounting evidence places these seemingly wasteful reactions at the fulcrum between metabolic signaling stress adaptation in bacteria. Specifically, pathways may mediate reprogramming observed when cells encounter growth-limiting constraints (i.e., nutrient limitation). Recent insights spotlight microbial an intricate network coordinates physiological programming energy conditions. Such intracellular cross stalk is pivotal survival competitive, fluctuating environments bacteria frequently nature. In light this paradigm signaling, are increasingly recognized strategies enable rewiring response stress. Overflow cycles generate secondary metabolites properties, alter flux rate acquisition, stimulate nodes trigger specific programs environmental challenges. Furthermore, observation such expensive under laboratory conditions purported be “energy limiting” fact suggest sufficiency, compelling us rethink how we model limitation starvation for

Language: Английский

---

Novel sanitization approach based on synergistic action of UV-A light and benzoic acid: Inactivation mechanism and a potential application in washing fresh produce

Food Microbiology, Journal Year: 2017, Volume and Issue: 72, P. 39 - 54

Published: Nov. 7, 2017

Language: Английский

---

The Potential Virulence Factors ofProvidencia stuartii: Motility, Adherence, and Invasion

BioMed Research International, Journal Year: 2018, Volume and Issue: 2018, P. 1 - 8

Published: Jan. 1, 2018

Providencia stuartii is the most common species capable of causing human infections. Currently P. involved in high incidence urinary tract infections catheterized patients. The ability bacteria to swarm on semisolid (viscous) surfaces and adhere invade host cells determines specificity disease pathogenesis its therapy. In present study we demonstrated morphological changes NK during migration viscous medium discussed adhesive invasive properties utilizing HeLa-M cell line as a model. To visualize interaction bacterial with eukaryotic vitro scanning electron confocal microscopy were performed. We found that are able epithelial these depend age culture. Also, infectious dose essential. microphotographs indicate after incubation together both adhered onto invaded into cells.

Language: Английский

---

Interactions between plant‐beneficial microorganisms in a consortium: Streptomyces microflavus and Trichoderma harzianum

Microbial Biotechnology, Journal Year: 2023, Volume and Issue: 16(12), P. 2292 - 2312

Published: July 18, 2023

Abstract The construction of microbial consortia is challenging due to many variables be controlled, including the cross‐compatibility selected strains and their additive or synergistic effects on plants. In this work, we investigated interactions in vitro, planta, at molecular level two elite biological control agents (BCAs), that Streptomyces microflavus strain AtB‐42 Trichoderma harzianum M10, understand attitude cooperate a consortium. observed strong cross‐antagonism between M10 agar plates diffusible metabolites volatile organic compounds. liquid co‐cultures, hindered growth very likely because secondary competition for nutrients. interaction co‐culture induced extensive transcriptional reprogramming both strains, especially pathways related ribosomes, protein synthesis, oxidoreductase activity, suggesting each recognized counterpart activated its defence responses. metabolome was also significantly affected. contrast, soil, partially contrasted by AtB‐42. roots tomato seedlings inoculated with consortium appeared smaller than single‐strain‐inoculated plants, indicating plants diverted some energy from development activation, as evidenced leaf transcriptome. stronger change compared single inoculants, demonstrated higher number differentially expressed genes. Although exerted effect inducing resistance responses inoculants. Our observations pose question usefulness sole vitro assays selecting BCAs construct vivo experiments should preferred, transcriptomics may greatly help elucidate activity beyond phenotypic plant.

Language: Английский

---

The proteome of bacterial membrane vesicles in Escherichia coli—a time course comparison study in two different media

Frontiers in Microbiology, Journal Year: 2024, Volume and Issue: 15

Published: March 6, 2024

Introduction Bacteria inhabit the in- and outside of human body, such as skin, gut or oral cavity where they play an innoxious, beneficial even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular (EVs). Several studies indicate bacterial (bMVs) a crucial role in microbiome-host interactions. However, composition bMVs their functionality under different culture conditions are still largely unknown. Methods To gain better insight into bMVs, we investigated E. coli (DSM 105380) from media Lysogeny broth (LB) RPMI 1640 throughout phases growth (lag-, log- stationary-phase). three time points (8 h, 54 168 h) two (LB 1640) were isolated by ultracentrifugation analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission (TEM) mass spectrometry-based proteomics (LC–MS/MS). Furthermore, examined pro-inflammatory cytokines IL-1β IL-8 monocyte cell line THP-1 upon bMV treatment. Results Particle numbers increased inoculation periods. The morphologies Cryo-EM/TEM similar at each point condition. Using proteomics, identified 140 proteins, common markers OmpA GroEL, present both all points. Additionally, able to detect growth-condition-specific proteins. Treatment six groups lead significantly high expressions. Conclusion Our study showed choice medium duration culturing influence protein composition. TEM/Cryo-EM results demonstrated presence intact bMVs. Common including OmpA, ribosome consistently be across tested conditions. our functional assays imply retain function result comparable cytokine induction.

Language: Английский

---