Enhancing bacterial cellulose production with hypoxia-inducible factors.

Komagataeibacter xylinus is an aerobic strain that produces bacterial cellulose (BC). Oxygen levels play a critical role in regulating BC synthesis in K. xylinus, and an increase in oxygen tension generally means a decrease in BC production. Fumarate nitrate reduction protein (FNR) and aerobic respiration control protein A (ArcA) are hypoxia-inducible factors, which can signal whether oxygen is present in the environment. In this study, FNR and ArcA were used to enhance the efficiency of oxygen signaling in K. xylinus, and globally regulate the transcription of the genome to cope with hypoxic conditions, with the goal of improving growth and BC production. FNR and ArcA were individually overexpressed in K. xylinus, and the engineered strains were cultivated under different oxygen tensions to explore how their overexpression affects cellular metabolism and regulation. Although FNR overexpression did not improve BC production, ArcA overexpression increased BC production by 24.0% and 37.5% as compared to the control under oxygen tensions of 15% and 40%, respectively. Transcriptome analysis showed that FNR and ArcA overexpression changed the way K. xylinus coped with oxygen tension changes, and that both FNR and ArcA overexpression enhanced the BC synthesis pathway. The results of this study provide a new perspective on the effect of oxygen signaling on growth and BC production in K. xylinus and suggest a promising strategy for enhancing BC production through metabolic engineering. KEY POINTS: • K. xylinus BC production increased after overexpression of ArcA • The young's modulus is enhanced by the ArcA overexpression • ArcA and FNR overexpression changed how cells coped with changes in oxygen tension.

Tailoring bacterial cellulose structure through CRISPR interference-mediated downregulation of galU in Komagataeibacter xylinus CGMCC 2955.

Diverse applications of bacterial cellulose (BC) have different requirements in terms of its structural characteristics. culturing Komagataeibacter xylinus CGMCC 2955, BC structure changes with alterations in oxygen tension. Here, the K. xylinus CGMCC 2955 transcriptome was analyzed under different oxygen tensions. Transcriptome and genome analysis indicated that BC structure is related to the rate of BC synthesis and cell growth, and galU is an essential gene that controls the carbon metabolic flux between the BC synthesis pathway and the pentose phosphate (PP) pathway. The CRISPR interference (CRISPRi) system was utilized in K. xylinus CGMCC 2955 to control the expression levels of galU. By overexpressing galU and interfering with different sites of galU sequences using CRISPRi, we obtained strains with varying expression levels of galU (3.20-3014.84%). By testing the characteristics of BC, we found that the porosity of BC (range: 62.99-90.66%) was negative with galU expression levels. However, the crystallinity of BC (range: 56.25-85.99%) was positive with galU expression levels; galU expression levels in engineered strains were lower than those in the control strains. Herein, we propose a new method for regulating the structure of BC to provide a theoretical basis for its application in different fields.

Identification of Quorum-Sensing Molecules of N-Acyl-Homoserine Lactone in Gluconacetobacter Strains by Liquid Chromatography-Tandem Mass Spectrometry.

Many Gram-negative bacteria can regulate gene expression in a cell density-dependent manner via quorum-sensing systems using N-acyl-homoserine lactones (AHLs), which are typical quorum-sensing signaling molecules, and thus modulate physiological characteristics. N-acyl-homoserine lactones are small chemical molecules produced at low concentrations by bacteria and are, therefore, difficult to detect. Here, a biosensor system method and liquid chromatography-tandem mass spectrometry were combined to detect and assay AHL production. As demonstrated by liquid chromatography-tandem mass spectrometry, Gluconacetobacter xylinus CGMCC No. 2955, a Gram-negative acetic acid-producing bacterium and a typical bacterial cellulose (BC) biosynthesis strain, produces six different AHLs, including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. Gluconacetobacter sp. strain SX-1, another Gram-negative acetic acid-producing bacterium, which can synthesize BC, produces seven different AHLs including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. These results lay the foundation for investigating the relationship between BC biosynthesis and quorum-sensing systems.

Preparation and characterization of antibacterial bacterial cellulose/chitosan hydrogels impregnated with silver sulfadiazine.

Hydrogels with pH sensitivity and stable mechanical and antibacterial properties have many desirable qualities and broad applications. A hydrogel based on bacterial cellulose and chitosan, impregnated with silver sulfadiazine (<1% w/w), was prepared using glutaraldehyde as the crosslinking agent. The presence of SSd was confirmed by Fourier transform infrared spectroscopy. Micropore size, swelling ratio, pH- sensitivity, and gram positive and negative antibacterial properties were studied by disk diffusion and colony forming unit. X-ray diffraction confirmed the presence of amorphous and crystalline regions in the hydrogel matrix following addition of SSd. The elemental composition, morphology, and mechanical properties of the hydrogels were characterized. Incorporation of SSd into bacterial cellulose-chitosan hydrogels significantly improved their mechanical and antibacterial properties. The antibacterial activity against E. coli and S. aureus was evaluated and SSd-BC/Ch hydrogels are more toxic to S. aureus than to E. coli. We use FESEM to observe bacterial morphology before and after exposure to SSd-BC/Ch hydrogels. The BacLight LIVE/DEAD membrane permeability kit is used to evaluate the membrane permeability of bacteria. These antibacterial hydrogels have many promising applications in food packaging, tissue engineering, drug delivery, clinical, biotechnological, and biomedical fields.

Bacterial cellulose and its potential for biomedical applications.

Bacterial cellulose (BC) is an important polysaccharide synthesized by some bacterial species under specific culture conditions, which presents several remarkable features such as microporosity, high water holding capacity, good mechanical properties and good biocompatibility, making it a potential biomaterial for medical applications. Since its discovery, BC has been used for wound dressing, drug delivery, artificial blood vessels, bone tissue engineering, and so forth. Additionally, BC can be simply manipulated to form its derivatives or composites with enhanced physicochemical and functional properties. Several polymers, carbon-based nanomaterials, and metal nanoparticles (NPs) have been introduced into BC by ex situ and in situ methods to design hybrid materials with enhanced functional properties. This review provides comprehensive knowledge and highlights recent advances in BC production strategies, its structural features, various in situ and ex situ modification techniques, and its potential for biomedical applications.