Thickness of Polyelectrolyte Layers of Separately Confined Bacteria Alters Key Physiological Parameters on a Single Cell Level.

Iaroslav Rybkin,Gleb Sukhorukov,Aleš Lapanje,Dmitry Gorin

doi:10.3389/fbioe.2019.00378

Abstract

Confinement of bacterial cells in a matrix or in capsules is an integral part of many biotechnological applications. Here, the well-known layer-by-layer method of deposition of a polyelectrolyte film a few nanometers in thickness to confine separated bacterial cells in permeable and physically durable shells has been examined. Due to the physical properties of such a confinement, we found that this method enables investigation of effects of physical barriers against mass gain and cell division. Using the method of time-lapse confocal microscopy, we observed a prolonged lag phase, dependent on the number of polyelectrolyte layers. In the confinement, both the GFP fluorescent signal from the leaking T7 promoter and the cell size were increased by factors of more than five and two, respectively. This creates a paradigm shift that enables use of mechanical entrapment for control of bacterial cell physiology and opens possibilities of controlling the division rate as well as gene expression. These effects can be attributed to the perturbation of the sensing of the cell size, which results in disproportional synthesis of a cell envelope impinging the intracellular material and compels cells to grow rapidly. In addition, the charged surface of cells enables prolonged intercellular physical interaction and results in spherically shaped microcolonies.

Highlights

Confinement of bacterial cells in matrices or capsules is an integral part of many applications in biotechnology
This approach of electrostatic modification of the bacterial surface has resulted in better adherence of probiotic bacteria to the surface of gut epithelia (Anselmo et al, 2016) and when cells are made in an LBL manner they can enhance the efficiency of vaccination due to the better presentation of the vaccine to immune cells (Speth et al, 2016)
By analysis of the growth curves on the population level we observed a significantly prolonged lag phase (Figure 1A) for cells on which we deposited more than one PE layer

Summary

Introduction

Confinement of bacterial cells in matrices or capsules is an integral part of many applications in biotechnology. Using well-established matrix entrapment methods the induction of fermentation activities, the ability to externally induce expression of specific proteins, for example green fluorescent protein (GFP) and the germination of spores have been observed and it was shown that it causes the “skin effect” of encapsulated cells. It is speculated, but not further investigated, that the skin effect is attributable to the increased permeability of the cell lining as a response to the less permeable capsule wall. It has been shown that confinement induces quorum sensing genes to reach characteristic cell densities sometimes even at the single cell level (Carnes et al, 2010)

Methods

Results

Conclusion