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...

Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.