Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

Abstract

Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (⌬dsbA and ⌬dsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells. Flocculation is a microbial behavior that can be applied in industrial fermentation and wastewater treatment. To date, numerous microorganisms have been studied to show their flocforming capabilities (1). In 1876, Louis Pasteur was the first to report flocculation, using the yeast Levure casseeuese (1). Later, Nakamura et al. confirmed flocculation in 19 other diverse microorganisms, including fungi, bacteria, and yeasts (2). In terms of bioapplication, Morimura et al. established a process for highperformance ethanol production from molasses by using flocculating Saccharomyces cerevisiae cells (3). Using flocculating yeast eliminates the costly centrifugation step required for cell recovery during continuous ethanol fermentation. Although the complete mechanism of microbial flocculation remains unclear, exopolymeric materials play a key role (1). In activated sludge, the components of the flocs typically include polysaccharides, proteins, and polynucleotides, and in pure laboratory cultures, many bacterial flocs are susceptible to cellulases, proteases, and DNases (4).As a common laboratory strain, Escherichia coli has a broad range of applications, and recent advancements in genetic engineering have shown its potential in the synthesis of various useful compounds. To date, many studies have attempted to induce E. coli flocculation by adding artificial flocculants composed of aluminum-based inorganic compounds or chitosan-based cationic polymers (5, 6). However, using such substances leads to severe damage to or the death of E. coli cells because of the forced flocculation. We previously demonstrated the self-generated flocculation of E. coli cells by overexpressing the native bc...

Displaying a recombinant protein on flocs self-produced by Escherichia coli through fused expression with elongation factor Ts

“…Considering that the flocs were washed several times with saline solution before observation, it can be speculated that the E. coli cells are firmly bound to the flocs. These results also support our previous study indicating that flocs mainly contain polymer fibers rather than cell aggregates [8]. As expected, flocs formed by BW25113/bcsB cells did not show any green fluorescence because of the lack of GFP introduction ( Fig.…”

“…In our previous study, we confirmed floc formation of E. coli BW25113/bcsB cells [8]. In this study, we also observed the flocculation of E. coli BW25113/bcsB/gfp and BW25113/bcsB/tsf-gfp cells (Fig.…”

“…E. coli K-12 strain BW25113 was obtained from the National BioResource Project (National Institute of Genetics (NIG), Mishima, Japan) [10]. The pNTR-SD-bcsB plasmid [11] was provided by NIG and the transformant was named BW25113/bcsB that forms spontaneous flocs [8]. pCA24N and ASKA-tsf-gfp plasmids were also provided by NIG [12].…”

“…We have previously reported self-produced flocculation of E. coli cells by overexpressing the native bcsB gene [6] that encodes a component of transmembrane cellulose synthase [7]. Further experiments clarified the spontaneous E. coli flocs were proteinous, and elongation factor Ts (Tsf) had the highest score among floc proteins in nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis [8]. Tsf is known to promote the release of GDP by forming an intermediate complex with another elongation factor, Tu, which is involved in the elongation cycle of protein biosynthesis [9].…”

Bacterial outer membrane vesicles: New insights and applications

“…A gram-negative facultative anaerobe, Shewanella oneidensis, shows hyper-vesiculation upon deletion of the degQ gene (Ojima et al, 2015). The degQ gene product is a periplasmic protease involved in the degradation of periplasmic proteins.…”