Enhancement of microbial biofilm formation by low antimicrobial doses is a critical problem in the medical field. The objective of this study was to propose a new drug candidate against the biofilm formation promoted by subinhibitory dose of antimicrobials. To determine the effect on biofilm formation of Escherichia coli, a subinhibitory concentration of lactoferrin (LF), a milk protein involved in a broad range of biological properties including antimicrobial action, or ampicillin (AMP), a typical antibiotic, was added to an E. coli cell culture in a 96-well microtiter plate. On the other hand, warfarin (WARF), an oral anticoagulant, or polymyxin B (PMB), a strong antibiotic for biofilm treatment, was added as an antagonist against the biofilm promoted by LF or AMP. The amount of biofilm formed at 100μg/mL LF in lysogeny broth medium was four times higher than in the absence of LF. Meanwhile, it was found that WARF suppressed the LF-promoted biofilm formation to a level comparable with the LF-free condition. WARF worked in a similar manner to PMB, which is known as an antibiofilm agent. Furthermore, WARF could also suppress the biofilm promoted by AMP. In conclusion, this study suggests that WARF can work as an antibiofilm agent against the biofilm formation promoted by subinhibitory dose of antimicrobials.

The utility of engineering flocculation is wildly recognized in applied and environmental microbiology. We previously reported self-produced flocculation of Escherichia coli cells by overexpressing the native bcsB gene that encodes a component of the cellulose synthesis pathway. Further experiments clarified that the spontaneous E. coli flocs were proteinous, and elongation factor Ts (Tsf) was the main component. In this study, we demonstrated successful expression of a fusion protein consisting of Tsf and green fluorescence protein (GFP) on E. coli flocs. Interestingly, the percentage of Tsf-GFP in total floc protein reached approximately 15% (w/w). The proposed design of a fusion protein with Tsf enables displaying a recombinant target protein on the floc structure.

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.