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Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

ABSTRACT: Bacterial cultivation techniques are classic, basic, and common processes used to characterize the physiological activity of bacteria in their environment. Owing to recent advances in bacterial cultivation techniques, the physiological activity of bacteria can be elucidated at the single-cell culture level. Here, we report a novel method to monitor the real-time activity of bacterial growth at the single-cell level inside giant unilamellar vesicles (GUVs). This method consists of two steps: 1)?encapsulation of single bacteria in 1-33?pL scale GUVs and 2)?immobilization of the GUVs on a planar lipid bilayer membrane on a glass surface. We directly observed single E. coli cells actively growing to a great number of cells inside GUVs. GUVs also protected the bacteria from external antibiotic compounds during prolonged cultivation for more than 24?h. This approach can be applied widely in the fields of biochemistry, biotechnology, microbiology, and synthetic biology.

SUBMITTER: Morita M

PROVIDER: S-EPMC6208190 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

Morita Masamune M Katoh Kaoru K Noda Naohiro N

ChemistryOpen 20180817 11

Bacterial cultivation techniques are classic, basic, and common processes used to characterize the physiological activity of bacteria in their environment. Owing to recent advances in bacterial cultivation techniques, the physiological activity of bacteria can be elucidated at the single-cell culture level. Here, we report a novel method to monitor the real-time activity of bacterial growth at the single-cell level inside giant unilamellar vesicles (GUVs). This method consists of two steps: 1) e ...[more]

PMID: 30402373

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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

Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

Publications

Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

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