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"Basicles": Microbial Growth and Production Monitoring in Giant Lipid Vesicles.

ABSTRACT: We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a riboflavin-producing bacterial strain. We show that stimulation as well as inhibition of bacterial production can be performed through the liposomal membrane simply by passive diffusion of inducing or antibiotic compounds, respectively. The possibility to introduce as well as accumulate compounds in liposomal cultivation compartments represents great advantage over the current state of the art systems, emulsion droplets, and gel beads. Additionally, the encapsulation of bacteria and monitoring of individual lipid vesicles have been accomplished on a single microfluidic device. The presented system paves the way toward highly parallel microbial cultivation and monitoring as required in biotechnology, basic research, or drug discovery.

SUBMITTER: Juskova P

PROVIDER: S-EPMC7462352 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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"Basicles": Microbial Growth and Production Monitoring in Giant Lipid Vesicles.

Jusková Petra P Schmid Yannick R F YRF Stucki Ariane A Schmitt Steven S Held Martin M Dittrich Petra S PS

ACS applied materials & interfaces 20190910 38

We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a ...[more]

PMID: 31454223

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Project description:BackgroundCystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli.ResultsUsing transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles.ConclusionsOur results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.

Project description:We developed a new (to our knowledge) protocol to generate giant unilamellar vesicles (GUVs) composed of mixtures of single lipopolysaccharide (LPS) species and Escherichia coli polar lipid extracts. Four different LPSs that differed in the size of the polar headgroup (i.e., LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles at physiological salt and pH conditions. Analysis of LPS incorporation into the membrane models (both oligolamellar vesicles and GUVs) shows that the final concentration of LPS is lower than that expected from the initial E. coli lipids/LPS mixture. In particular, our protocol allows incorporation of no more than 15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for LPS-Rc and LPS-Rd (with respect to total lipids). We used the GUVs to evaluate the impact of different LPS species on the lateral structure of the host membrane (i.e., E. coli polar lipid extract). Rhodamine-DPPE-labeled GUVs show the presence of elongated micrometer-sized lipid domains for GUVs containing either LPS-Rc or LPS-Rd above 10 mol %. Laurdan GP images confirm this finding and show that this particular lateral scenario corresponds to the coexistence of fluid disordered and gel (LPS-enriched)-like micron-sized domains, in similarity to what is observed when LPS is replaced with lipid A. For LPSs containing the more bulky polar headgroup (i.e., LPS-smooth and LPS-Ra), an absence of micrometer-sized domains is observed for all LPS concentrations explored in the GUVs (up to ?15 mol %). However, fluorescence correlation spectroscopy (using fluorescently labeled LPS) and Laurdan GP experiments in these microscopically homogeneous membranes suggests the presence of LPS clusters with dimensions below our microscope's resolution (?380 nm radial). Our results indicate that LPSs can cluster into gel-like domains in these bacterial model membranes, and that the size of these domains depends on the chemical structure and concentration of the LPSs.

Dataset Information

"Basicles": Microbial Growth and Production Monitoring in Giant Lipid Vesicles.

Publications

"Basicles": Microbial Growth and Production Monitoring in Giant Lipid Vesicles.

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