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High production cost is a significant barrier to commercial bacterial nanocellulose (BNC) production. This study addresses this issue using a low-cost molasses and cheese whey medium via Gluconacetobacter hansenii. The one-factor-at-a-time method investigated the effect of critical factors on BNC production, including total sugar and total protein concentrations (g/L), initial pH, and additives such as ethanol and acetic acid (%(v/v)). The productivity in the HS medium was 0.125 g/L/day, while the low-cost medium without additives achieved a productivity of 0.5275 g/L/day. Although the addition of ethanol decreased the productivity, the inclusion of 0.4 %(v/v) acetic acid increased the productivity to 0.64 g/L/day. Using the low-cost medium with acetic acid led to a 40-fold reduction in production costs compared to the HS medium. Furthermore, transitioning from HS media to the low-cost medium resulted in BNC with thicker fibres, a higher crystallinity index (%) and improved mechanical properties. The ratio of I α / I β in BNC produced in HS media decreased from 1.68 to 1.09 in the low-cost medium. The thermal properties of BNC produced in the low-cost medium also showed slight improvements compared to those in the HS medium. The degree of polymerization significantly increased from 1096 to 1457 in the low-cost medium compared to HS media. These findings highlight the potential of using a low-cost medium to produce BNC with enhanced properties, offering a sustainable and cost-effective alternative to conventional plastics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The current study intended to analyze the impact of ethanol and lactic acid on the bacterial cellulose yield as well as physicochemical and mechanical properties, by using Gluconacetobacter kombuchae . The optimization of ethanol and lactic acid concentration has been done by using one-way ANOVA. Both the supplements significantly enhance the yield of bacterial cellulose (BC) as compared to the standard Hestrin-Schramm medium (control). Optimization leads to significant increase in BC yield as compared to the control, i.e., the addition, of optimized concentration of lactic acid (0.6%) increases the yield from (0.78 ± 0.026) g to (4.89 ± 0.020) g dry weight, and optimized concentration of ethanol (1%) increases the yield from (0.73 ± 0.057) g to (3.7 ± 0.01) g dry weight. Various physicochemical and mechanical properties of BC films produced in different media (i.e., HS, HS + Ethanol, and HS + Lactic acid), such as the crystallinity, structure, tensile strength, strain at break, Young's modulus, and water holding capacity, were also examined, by employing various techniques such as SEM, FTIR, XRD, etc. BC produced in medium supplemented with the optimum concentration of both the additives were found to possesses higher porosity. Though, slight decline in crystallinity was observed. But the tensile strength and strain at break, were upgraded 1.5-2.5 times, 2-2.5 times, respectively. This article attempted to present a method for enhancing BC yields and characteristics that may lead to more widespread and cost-effective use of this biopolymer.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19 th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing β- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9 g/L., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The objective of the work is to examine the potential utilization of Palmyra palm jaggery (PPJ) for the enhancement of bacterial cellulose (BC) production by Gluconacetobacter liquefaciens. To evaluate the culturing condition, the production of BC fermentation was carried out in batch mode using different carbon sources namely glucose, sucrose and PPJ. PPJ in the HS medium (PHS medium) resulted maximum concentration of BC (14.35 ± 0.18 g/L) under shaking condition than other carbon sources in HS medium. The influence of different medium variables including initial pH and nitrogen sources on BC production was investigated using PHS medium under shaking condition. The maximum BC concentration of 17.79 ± 2.4 g/L was obtained in shaking condition at an initial pH of 5.6 using yeast extract as nitrogen source. Stoichiometric equation for the cell growth and BC synthesis was developed using elemental balance approach. The metabolic heat of reaction (40 kcal generated per liter of medium) was evaluated using electron balance approach. Based on the process economic analysis and the yield of BC during the fermentation, PHS medium without nitrogen source could be a promising cost-effective nutrient than HS medium. Thermal stability, crystallinity index and structural characterizations of produced BC using PPJ medium were evaluated using TGA, XRD and FTIR and the obtained results were compared with HS medium containing glucose and sucrose., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cadmium (Cd) is a heavy metal used as raw material for several fertilizers and pesticides. The increase of Cd concentration in soils has been observed in cultivated areas, affecting animals, plants, and microorganisms. Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium able to survive under adverse environmental conditions. Here, we investigated key mechanisms involved with the resistance of G. diazotrophicus to Cd. Proteomic analyses revealed that the main pathways regulated in response to Cd are nutrient uptake, multidrug efflux pumps, response to oxidative stress, and protein quality control system. Extracytoplasmic proteins related to multidrug efflux pumps were up-accumulated, while several proteins related to nutrients uptake were down-accumulated. The relevance of these pathways for bacterial resistance to Cd was investigated by reverse genetic analysis using mutants defective for nutrient uptake (tdbr, ompW, and oprB), multidrug efflux (czcC), response to oxidative stress (ggt), and protein quality control system (clpX). Our data demonstrated the essential role of the tdbr and czcC genes for resistance to Cd in G. diazotrophicus. These results contribute to a better understanding of the resistance mechanisms to Cd in G. diazotrophicus, shedding light on responses associated with extracytoplasmic compartments., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Bacterial cellulose (BC) has excellent material properties and can be produced sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli ( E. coli ). Here, a simple approach is reported for producing highly programmable BC materials through incorporation of engineered E. coli . The acetic acid bacterium Gluconacetobacter hansenii is cocultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which are then colonized by the E. coli upon transfer to selective lysogeny broth media. It is shown that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, capsules are produced which can alter their own bulk physical properties through enzyme-induced biomineralization. This novel system uses a simple fabrication process, based on the autonomous activity of two bacteria, to significantly expand the functionality of BC-based living materials., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity. KEY POINTS: • AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5. • Acetaldehyde accumulated from ethanol in the absence of AldFGH. • Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength, and stiffness prevent dehydration and mechanical disruption of the biofilm. Bacteria in the genus Gluconacetobacter secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused-ion-beam milling of native bacterial biofilms to image cellulose-synthesizing Gluconacetobacter hansenii and Gluconacetobacter xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several micrometers in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, which we have named the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We found that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line to form higher-order cellulose structures, such as sheets and ribbons. IMPORTANCE This work's relevance for the microbiology community is twofold. It delivers for the first time high-resolution near-native snapshots of Gluconacetobacter spp. (previously Komagataeibacter spp.) in the process of cellulose ribbon synthesis, in their native biofilm environment. It puts forward a noncharacterized cytoskeleton element associated with the side of the cell where the cellulose synthesis occurs. This represents a step forward in the understanding of the cell-guided process of crystalline cellulose synthesis, studied specifically in the Gluconacetobacter genus and still not fully understood. Additionally, our successful attempt to use cryo-focused-ion-beam milling through biofilms to image the cells in their native environment will drive the community to use this tool for the morphological characterization of other studied biofilms., (Copyright © 2021 Nicolas et al.)

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N 2 ) to ammonia (NH 3 ). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN's protection mechanism involves decreasing the binding affinity of CO to nitrogenase's active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase., Competing Interests: Conflict of interest The authors declare no conflict of interest with the content of the article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Probiotic Gluconacetobacter strains are intestinal microbes with beneficial effects on human health. Recently, researchers have used these strains to biosynthesize metal and non-metal nanoparticles for treating various chronic diseases. Despite their importance in nanotechnology, gold nanoparticles (AuNPs) biosynthesized by Gluconacetobacter species have not been clearly identified for treating inflammation and inflammation-associated diseases. While ginsenoside CK has strong pharmaceutical activity, it also has strong cytotoxicity and hydrophobicity which is hurdle to make formulation. Peptide-nanoparticle hybrids are gaining increasing attention for their potential biomedical applications, including human inflammatory diseases. Herein, we developed peptide CopA3 surface conjugated and ginsenoside compound K (CK) loaded gold nanoparticles (GNP-CK-CopA3), which intracellularly synthesised by the probiotic Gluconacetobacter liquefaciens kh-1, to target lipopolysaccharide (LPS)-activated RAW264.7 macrophages. The synthetic GNP-CK-CopA3 was characterised by various instrumental techniques. The results of our cellular uptake and MTT assays exhibited obvious drug intracellular delivery without significant cytotoxicity. In addition, pre-treatment with GNP-CK-CopA3 significantly ameliorated LPS-induced nitric oxide (NO) and reactive oxygen species (ROS) production and suppressed the mRNA and protein expression of pro-inflammatory cytokines in macrophages. Furthermore, GNP-CK-CopA3 efficiently inhibited the activation of the nuclear factor-κB (NF-κB) and mitogen-activating protein kinase (MAPK) signalling pathways. Taken together, our findings highlight the potential of using peptide-nanoparticle hybrids in the development of anti-inflammatory approaches and providing the experimental foundation for further application.

Acetic acid bacteria form a complex microbiota that plays a fundamental role in the industrial production of vinegar through the incomplete oxidation reaction from ethanol to acetic acid. The organoleptic properties and the quality of vinegar are influenced by many factors, especially by the raw material used as acetification substrate, the microbial diversity and the technical methods employed in its production. The metaproteomics has been considered, among the new methods employed for the investigation of microbial communities, since it may provide information about the microbial biodiversity and behaviour by means of a protein content analysis. In this work, alcohol wine vinegar was produced through a submerged culture of acetic acid bacteria using a pilot acetator, operated in a semi-continuous mode, where the main system variables were monitored and the cycle profile throughout the acetification was obtained. Through a first approach, at qualitative level, of a metaproteomic analysis performed at relevant moments of the acetification cycle (end of fast and discontinuous loading phases and just prior to unloading phase), it is aimed to investigate the microbiota existent in alcohol wine vinegar as well as its changes during the cycle; to our knowledge, this is the first metaproteomics report carried out in this way on this system. A total of 1723 proteins from 30 different genera were identified; 1615 out of 1723 proteins (93.73%) belonged to the four most frequent (%) genera: Acetobacter, Gluconacetobacter, Gluconobacter and Komagataeibacter. Around 80% of identified proteins belonged to the species Komagataeibacter europaeus. In addition, GO Term enrichment analysis highlighted the important role of catalytic activity, organic cyclic compound binding, metabolic and biosynthesis processes throughout acetic acid fermentation. These findings provide the first step to obtain an AAB profile at omics level related to the environmental changes produced during the typical semi-continuous cycles used in this process and it would contribute to the optimization of operating conditions and improving the industrial production of vinegar., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this paper, we perform a systematic analysis of the structural organization of bacterial cellulose (BC). We report four types of organization of the BC mass, produced by Gluconacetobacter hansenii that occur depending on cultivation conditions. Two of those, particularly, plywood type one and layers of micro-sized tubes were observed and described for the first time. In spherical BC particles (pellets), we found the layered structure that had previously been reported for planar geometry only. We suggest a model explaining why layers form in BC films and attempt to reveal the impact of different factors on the BC microscale morphology. We assume that the main factor that has direct impact on the type of structure formed is the rate of BC mass accumulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest, (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1 -ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13 . The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1 -ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro.

In this work, we report a convenient method of grafting non-leachable bioactive amine functions onto the surface of bacterial cellulose (BC) nanofibrils, via a simple silylation treatment in water. Two different silylation protocols, involving different solvents and post-treatments were envisaged and compared, using 3-aminopropyl-trimethoxysilane (APS) and (2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPS) as silylating agents. In aqueous and controlled conditions, water-leaching resistant amino functions could be successfully introduced into BC, via a simple freeze-drying process. The silylated material remained highly porous, hygroscopic and displayed sufficient thermal stability to support the sterilization treatments generally required in medical applications. The impact of the silylation treatment on the intrinsic anti-bacterial properties of BC was investigated against the growth of Escherichia coli and Staphylococcus aureus. The results obtained after the in vitro studies revealed a significant growth reduction of S. aureus within the material., (Copyright © 2019. Published by Elsevier Ltd.)

Including additives in the culture media during bacterial cellulose (BC) biosynthesis is a traditional method to produce BC-based nanocomposites. This study examines a novel fermentation process, which is to co-culture Gluconacetobacter hansenii (G. hansenii) with Escherichia coli (E. coli) under static conditions, to produce BC pellicles with enhanced mechanical properties. The mannose-rich exopolysaccharides (EPS) synthesized by E. coli were incorporated into the BC network and affected the aggregation of co-crystallized microfibrils without significantly changing the crystal sizes of BC. When co-culturing G. hansenii ATCC 23769 with E. coli ATCC 700728, which produced a low concentration of EPS at 3.3 ± 0.7 mg/L, the BC pellicles exhibited a Young's modulus of 4,874 ± 1144 MPa and a stress at break of 80.7 ± 21.1 MPa, which are 81.9% and 79.3% higher than those of pure BC, respectively. The growth dynamics of the two co-cultured strains suggested that the production of BC and EPS were enhanced through co-culturing fermentation., (Copyright © 2019. Published by Elsevier Ltd.)

The biosynthesis of antioxidant pigments, namely, betalains, was believed to be restricted to Caryophyllales plants. This paper changes this paradigm, and enzyme mining from bacterial hosts promoted the discovery of bacterial cultures producing betalains. The spectrum of possible sources of betalain pigments in nature is broadened by our description of the first betalain-forming bacterium, Gluconacetobacter diazotrophicus The enzyme-specific step is the extradiol cleavage of the precursor amino acid l-dihydroxyphenylalanine (l-DOPA) to form the structural unit betalamic acid. Molecular and functional work conducted led to the characterization of a novel dioxygenase, a polypeptide of 17.8 kDa with a K m of 1.36 mM, with higher activity and affinity than those of its plant counterparts. Its superior activity allowed the first experimental characterization of the early steps in the biosynthesis of betalains by fully characterizing the presence and time evolution of 2,3- and 4,5-seco-DOPA intermediates. Furthermore, spontaneous chemical reactions are characterized and incorporated into a comprehensive enzymatic-chemical mechanism that yields the final pigments. IMPORTANCE Several studies have demonstrated the health-promoting effects of betalains due to their high antioxidant capacity and their positive effect on the dose-dependent inhibition of cancer cells and their proliferation. To date, betalains were restricted to plants of the order Caryophyllales and some species of fungi, but the present study reveals the first betalain-producing bacterium, as well as the first steps in the formation of pigments. This finding demonstrates that betalain biosynthesis can be expanded to prokaryotes., (Copyright © 2019 Contreras-Llano et al.)

Bacterial cellulose (BC) has been gaining importance over the past decades as a versatile material that finds applications in diverse industries. However, a secured supply is hindered by the slow production rate and batch-to-batch variability of the yield. Here, we report a rational approach for characterising the BC production process using Design of Experiment (DoE) methodology to study the impact of different parameters on desired process attributes. Notably, we found that the carbon source used for bacterial growth significantly impacts the interplay between the process variables and affects the desired outcomes. We therefore, propose that the highest priority process outcome in this study, the yield, is a function of the carbon source and optimal reactor design. Our systematic approach has achieved projected BC yields as high as ∼40 g/L for Gluconacetobacter hansenii 53582 grown on sucrose as the carbon source compared to the widely reported yields of ∼10 g/L., (Copyright © 2018. Published by Elsevier Ltd.)

Pecan nutshell is an abundant waste with a high content of carbohydrates. According to its chemical composition, pecan nutshell could be used as carbon source for Gluconacetobacter entanii, a bacterium that produces cellulose with high purity and nanometric characteristics. Bacterial cellulose (BC) was obtained from a static culture medium using pecan nutshell as carbon source and saccharose as control. Results showed that the pecan nutshell could be used as carbon source for production of BC. The cellulose yield ranged around 2.816 ± 0.040 g/L for 28 days. The morphological, structural and chemical properties of the cellulose produced were similar to those reported for others BC. The spectroscopic characterization indicated the chemical functionalization of BC and the reduction of its crystallinity. The production of BC with G. entanii using pecan nutshell as carbon source, is the first report. The BC could have potential use in chemical functionalization and in the preparation of biocomposites., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium that is used as a bioinoculant. Phosphate (Pi) modulates intracellular polyphosphate (polyP) levels in Escherichia coli, affecting cellular fitness and biofilm formation capacity. It currently remains unclear whether environmental Pi modulates polyP levels in G. diazotrophicus to enhance fitness in view of its technological applications. In high Pi media, cells accumulated polyP and degraded it, thereby improving survival, tolerance to environmental stressors, biofilm formation capacity on abiotic and biotic surfaces, and competence as a growth promoter of strawberry plants. The present results support the importance of Pi and intracellular polyP as signals involved in the survival of G. diazotrophicus.

To reduce the cost of obtaining bacterial cellulose, acidic by-products of the alcohol and dairy industries were used without any pretreatment or addition of other nitrogen sources. Studies have shown that the greatest accumulation of bacterial cellulose (6.19g/L) occurs on wheat thin stillage for 3 days of cultivation under dynamic conditions, which is almost 3 times higher than on standard Hestrin and Schramm medium (2.14g/L). The use of whey as a nutrient medium makes it possible to obtain 5.45g/L bacterial cellulose under similar conditions of cultivation. It is established that the pH of the medium during the growth of Gluconacetobacter sucrofermentans B-11267 depends on the feedstock used and its initial value. By culturing the bacterium on thin stillage and whey, there is a decrease in the acidity of the waste. It is shown that the infrared spectra of bacterial cellulose obtained in a variety of environments have a similar character, but we found differences in the micromorphology and crystallinity of the resulting biopolymer., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

This study aimed to investigate the effects of supplementation with bacterial cellulose (BC), konjac glucomannan (KGM) and combined BC/KGM fiber on high-fat (HF)-diet-induced obesity in C57BL/6J mice. The results showed that combined supplementation with BC/KGM in HF-fed mice was more efficient in reducing body weight, lowing serum lipid profiles and suppressing insulin resistance than single supplementation with BC or KGM. Moreover, supplementation with combined BC/KGM fiber more efficiently alleviated HF-diet-induced liver injury by decreasing hepatic steatosis in comparison with supplementation with BC or KGM alone. Furthermore, supplementation with combined BC/KGM fiber in HF-fed mice had a more positive effect on obesity-associated hepatic inflammation by reducing levels of TNF-α and IL-6 and suppressing the protein expression of Nrf-2/ARE in comparison with supplementation with BC or KGM alone. Consumption of these dietary fibers, especially mixed BC/KGM, resulted in an improved antioxidant defense system and reduced lipid peroxidation in the liver by increasing the activity of antioxidant enzymes and reducing the formation of MDA in the liver. Moreover, supplementation with these fibers regulated the levels of leptin and adiponectin and inhibited the protein expression of PPARγ by reducing the size of cells in the adipose tissue of HF diet-fed mice. Therefore, fiber supplementation (especially with combined BC/KGM) efficiently inhibited HF-induced obesity in mice by reducing insulin resistance, liver injury and inflammation, enhancing the antioxidant defense system and regulating the secretion of adipocytokines and adipogenesis-associated proteins.

Aims: Obtain varieties of Gluconacetobacter hansenii from original strain ATCC 23729 with greater efficiency to produce bacterial cellulose (BC) membrane with better dry mass yield for application as support of sustained antimicrobials' drug release., Methods and Results: Application of different chemical and physical conditions (pH, temperature and UV light exposure) to obtain different G. hansenii varieties with high capacity to produce BC membranes. Characterization of the G. hansenii variants was performed by scanning electron microscopy (SEM) and optical microscopy of the colony-forming units. BC membrane produced was characterized by SEM, infrared spectroscopy and X-ray diffraction. The BC produced by variants isolated after incubation at 35°C showed elevated dry mass yield and high capacity of retention and sustained release of ceftriaxone antibiotic with the produced BC by original G. hansenii ATCC 23769 strain subjected to incubation at 28°C and with commercial BC., Conclusion: The application of different chemical and physical conditions constitutes an important method to obtain varieties of micro-organisms with dissimilar metabolism advantageous in relation to the original strain in the BC production., Significance and Impact of the Study: These results demonstrate the importance of in vivo studies for the application, in medicine, of BC membranes as support for antimicrobial-sustained release for the skin wound treatment., (© 2018 The Society for Applied Microbiology.)

Recent research was conducted to evaluate the healing efficiency of bacterial cellulose (BC) as a wound dressing in different pHs and its possibility of being a smart wound dressing that can indicate pHs. BC was produced by environmentally isolated bacterial strains. After washing the best achieved BC, it was floated in normal saline with different pHs with phenol red used as a pH indicator. Finally the wound healing effects of the acidic, neutral and alkaline BC membranes were evaluated in rat cutaneous wounds. Results showed that one of the isolates which its partial 16srRNA genome had 95% similarity with Gluconacetobacter intermedius, had the thickest layer. The microscopic and macroscopic evaluations showed that the acidic BC had the best healing activity. Although the color of the films remained unchanged during the experiments because they were transparent and thin, these changes could not be easily seen. This suggests the use of thicker films such as the ones which are cross linked with some materials (e.g., sterile gauze). In conclusion the pH can affect the healing ability of natural BC and acidic pH had the best wound healing efficiency. In future it is better to use the acidic BC instead of natural one for different wound healing purposes.

Bacterial cellulose (BC) is a biocompatible material with versatile applications. However, its large-scale production is challenged by the limited biological knowledge of the bacteria. The advent of synthetic biology has lead the way to the development of BC producing microbes as a novel chassis. Hence, investigation on optimal growth conditions for BC production and understanding of the fundamental biological processes are imperative. In this study, we report a novel analytical platform that can be used for studying the biology and optimizing growth conditions of cellulose producing bacteria. The platform is based on surface growth pattern of the organism and allows us to confirm that cellulose fibrils produced by the bacteria play a pivotal role towards their chemotaxis. The platform efficiently determines the impacts of different growth conditions on cellulose production and is translatable to static culture conditions. The analytical platform provides a means for fundamental biological studies of bacteria chemotaxis as well as systematic approach towards rational design and development of scalable bioprocessing strategies for industrial production of bacterial cellulose.

Bacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccp Ax). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 °C, 30 °C, and 37 °C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000-3000 μm and a diameter of 10-20 μm, which were remarkably larger than the fibres of BC typically produced by G. hansenii.

The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus, the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S-adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the KeLrp variant in KGMA7203. KGMA7110 (0.69±0.27 μM) and KGMA7203 (4.90±0.61 μM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28±0.26 μM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that KeLrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In this work, for the first time bacterial cellulose (BC) hydrogel membranes were used for the fabrication of antimicrobial cellulosic nanocomposites by hydrothermal deposition of Cu derivative nanoparticles (i.e.Cu(0) and CuxOy species). BC-Cu nanocomposites were characterized by FTIR, SEM, AFM, XRD and TGA, to study the effect of hydrothermal processing time on the final physicochemical properties of final products. XRD result show that depending on heating time (3-48h), different CuxOy phases were achieved. SEM and AFM analyses unveil the presence of the Cu(0) and copper CuxOy nanoparticles over BC fibrils while the surface of 3D network became more compact and smother for longer heating times. Furthermore, the increase of heating time placed deleterious effect on the structure of BC network leading to decrease of BC crystallinity as well as of the on-set degradation temperature. Notwithstanding, BC-Cu nanocomposites showed excellent antimicrobial activity against E. coli, S. aureus and Salmonella bacteria suggesting potential applications as bactericidal films., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N 2 ) to ammonia (NH 3 ). In its most prevalent form, it consists of two proteins, the catalytic molybdenum-iron protein (MoFeP) and its specific reductase, the iron protein (FeP). A defining feature of nitrogenase is that electron and proton transfer processes linked to substrate reduction are synchronized by conformational changes driven by ATP-dependent FeP-MoFeP interactions. Yet, despite extensive crystallographic, spectroscopic, and biochemical information on nitrogenase, the structural basis of the ATP-dependent synchronization mechanism is not understood in detail. In this chapter, we summarize some of our efforts toward obtaining such an understanding. Experimental investigations of the structure-function relationships in nitrogenase are challenged by the fact that it cannot be readily expressed heterologously in nondiazotrophic bacteria, and the purification protocols for nitrogenase are only known for a small number of diazotrophic organisms. Here, we present methods for purifying and characterizing nitrogenase from a new model organism, Gluconacetobacter diazotrophicus. We also describe procedures for observing redox-dependent conformational changes in G. diazotrophicus nitrogenase by X-ray crystallography and electron paramagnetic resonance spectroscopy, which have provided new insights into the redox-dependent conformational gating processes in nitrogenase., (© 2018 Elsevier Inc. All rights reserved.)

Films with antioxidant activity were prepared by combining chitosan (CH) and epigallocatechin-3-gallate (EGCG) with nano-bacterial cellulose (BC) as a reinforcement agent. Results showed that BC addition improved the qualities of films jeopardized by EGCG. Fourier Transform Infrared (FTIR) spectroscopy analysis confirmed that there were intermolecular interactions among BC, CH and EGCG though hydrogen bonding. Consequently, BC addition increased the crystallinity and thermal stability of CH-BC5/10-EGCG15/30 films analyzed by X-ray diffraction (XRD) and thermogravimetric (TGA) analysis. BC addition also significantly decreased water solubility (WS), from 26.54% to 20.80%, increased tensile strength (TS), from 18.71 to 44.17MPa, and increased elongation at break (EAB), from 2.72% to 7.34%. Moreover, BC and EGCG had high affinity for each other, as BC addition lowered the release of EGCG from CH films with a higher ABTS radical scavenging ability. Therefore, BC can be used as a sustained release carrier of EGCG in antioxidant active films., (Copyright © 2017. Published by Elsevier Ltd.)

Nanofibrillated bacterial cellulose (NFBC) is produced by culturing a cellulose-producing bacterium (Gluconacetobacter intermedius NEDO-01) with rotation or agitation in medium supplemented with carboxymethylcellulose (CMC). Despite a high yield and dispersibility in water, the product immediately aggregates in organic solvents. To broaden its applicability, we prepared amphiphilic NFBC by culturing strain NEDO-01 in medium supplemented with hydroxyethylcellulose or hydroxypropylcellulose instead of CMC. Transmission electron microscopy analysis revealed that the resultant materials (HE-NFBC and HP-NFBC, respectively) comprised relatively uniform fibers with diameters of 33 ± 7 and 42 ± 8 nm, respectively. HP-NFBC was dispersible in polar organic solvents such as methanol, acetone, isopropyl alcohol, acetonitrile, tetrahydrofuran (THF), and dimethylformamide, and was also dispersible in poly(methyl methacrylate) (PMMA) by solvent mixing using THF. HP-NFBC/PMMA composite films were highly transparent and had a higher tensile strength than neat PMMA film. Thus, HP-NFBC has a broad range of applications, including as a filler material.

Bacterial cellulose (BC) is a homopolymer and it is distinguished from plant-based cellulose by its unique properties such as high purity, high crystallinity, high water-holding capacity, and good biocompatibility. Microalgae are unicellular, photosynthetic microorganisms and are known to have high protein, starch, and oil content. In this study, Chlorella vulgaris was evaluated as source of glucose for the production of BC. To increase the starch content of algae the effect of nutrient starvation (nitrogen and sulfur) and light deficiency were tested in a batch assay. The starch contents (%) were 5.27 ± 0.04, 7.14 ± 0.18, 5.00 ± 0.08, and 1.35 ± 0.04 for normal cultivation, nitrogen starvation, sulfur starvation, and dark cultivation conditions, respectively. The performance of enzymatic and acidic methods was compared for the starch hydrolysis. This study demonstrated for the first time that acid hydrolysate of algal starch can be used to substitute glucose in the fermentation medium of Komagataeibacter hansenii for BC production. Glucose was used as a control for BC production. BC production yields on dry weight basis were 1.104 ± 0.002 g/L and 1.202 ± 0.005 g/L from algae-based glucose and glucose, respectively. The characterization of both BCs produced from glucose and algae-based glucose was investigated by scanning electron microscopy and Fourier transform infrared spectroscopy. The results have shown that the structural characteristics of algae-based BC were comparable to those of glucose-based BC.

Isolate B17 from Kombucha was estimated to be an efficient producer of bacterial cellulose (BC). The isolate was deposited under the number P 1463 and identified as Komagataeibacter rhaeticus by comparing a generated amplified fragment length polymorphism (AFLP™) DNA fingerprint against a reference database. Static cultivation of the K. rhaeticus strain P 1463 in Hestrin and Schramm (HS) medium resulted in 4.40 ± 0.22 g/L BC being produced, corresponding to a BC yield from glucose of 25.30 ± 1.78 %, when the inoculum was made with a modified HS medium containing 10 g/L glucose. Fermentations for 5 days using media containing apple juice with analogous carbon source concentrations resulted in 4.77 ± 0.24 g/L BC being synthesised, corresponding to a yield from the consumed sugars (glucose, fructose and sucrose) of 37.00 ± 2.61 %. The capacity of K. rhaeticus strain P 1463 to synthesise BC was found to be much higher than that of two reference strains for cellulose production, Komagataeibacter xylinus DSM 46604 and Komagataeibacter hansenii DSM 5602 T , and was also considerably higher than that of K. hansenii strain B22, isolated from another Kombucha sample. The BC synthesised by K. rhaeticus strain P 1463 after 40 days of cultivation in HS medium with additional glucose supplemented to the cell culture during cultivation was shown to have a degree of polymerization of 3300.0 ± 122.1 glucose units, a tensile strength of 65.50 ± 3.27 MPa and a length at break of 16.50 ± 0.83 km. For the other strains, these properties did not exceed 25.60 ± 1.28 MPa and 15.20 ± 0.76 km.

Novel thin and smooth deuterated cellulose films were synthesised to visualize adsorbed bio-macromolecules using contrast variation neutron reflectivity (NR) measurements. Incorporation of varying degrees of deuteration into cellulose was achieved by growing Gluconacetobacter xylinus in deuterated glycerol as carbon source dissolved in growth media containing D 2 O. The derivative of deuterated cellulose was prepared by trimethylsilylation(TMS) in ionic liquid(1-butyl-3-methylimidazolium chloride). The TMS derivative was dissolved in toluene for thin film preparation by spin-coating. The resulting film was regenerated into deuterated cellulose by exposure to acidic vapour. A common enzyme, horseradish peroxidase (HRP), was adsorbed from solution onto the deuterated cellulose films and visualized by NR. The scattering length density contrast of the deuterated cellulose enabled accurate visualization and quantification of the adsorbed HRP, which would have been impossible to achieve with non-deuterated cellulose. The procedure described enables preparing deuterated cellulose films that allows differentiation of cellulose and non-deuterated bio-macromolecules using NR.

Bacterial cellulose (BC) exhibits unique properties including high mechanical strength and high crystallinity. Improvement in the mechanical properties of BC is sought for many applications ranging from food to structural composites to biomedical materials. In this study, different additives including carboxymethyl cellulose (CMC), pectin, gelatin, cornstarch, and corn steep liquor were included in the fermentation media to alter the BC produced. Three different concentrations (1%, 3% and 5%) were chosen for each of the additives, with no additive (0%) as the control. The produced BC was then analyzed to determine tensile and compression modulus. Amongst the tested additives, BC produced in media containing 3% (w/v) pectin had the maximum compressive modulus (142kPa), and BC produced in media containing 1% (w/v) gelatin exhibited the maximum tensile modulus (21MPa). Structural characteristics of BC and BC-additive composites were compared using X-Ray diffraction (XRD). The crystal size and crystallinity of BC was reduced when grown in the presence of CMC and gelatin while pectin only decreased the crystallite size. This suggested that CMC and gelatin may be incorporated into the BC fibril structure. The field emission scanning electron microscopy (FESEM) images showed the increased micro-fibril aggregation in BC pellicles grown in the presence of additives to the culture media., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus, and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids, and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system, and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar.

Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

The current study was intended to produce bio-cellulose through a cell-free system developed by disrupting Gluconacetobacter hansenii PJK through bead-beating. Microscopic analysis indicated the complete disruption of cells (2.6 × 10(7) cells/mL) in 20 min that added 95.12 μg/mL protein, 1.63 mM ATP, and 1.11 mM NADH into the medium. A liquid chromatography mass spectrometry/mass spectrometry linear trap quadrupole (LC-MS/MS LTQ) Orbitrap analysis of cell-lysate confirmed the presence of all key enzymes for bio-cellulose synthesis. Under static conditions at 30 °C, microbial and cell-free systems produced 3.78 and 3.72 g/L cellulose, corresponding to 39.62 and 57.68% yield, respectively after 15 days. The improved yield based on consumed glucose indicated the superiority of cell-free system. Based on current findings and literature, we hypothesized a synthetic pathway for bio-cellulose synthesis in the cell-free system. This approach can overcome some limitations of cellulose-producing cells and offers a wider scope for synthesizing cellulose composites with bactericidal elements through in situ synthesizing approaches., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Levansucrases are members of the glycoside hydrolase family and catalyse both the hydrolysis of the substrate sucrose and the transfer of fructosyl units to acceptor molecules. In the presence of sufficient sucrose, this may either lead to the production of fructooligosaccharides or fructose polymers. Aim of this study is to rationalise the differences in the polymerisation properties of bacterial levansucrases and in particular to identify structural features that determine different product spectrum in the levansucrase of the Gram-negative bacterium Erwinia amylovora (Ea Lsc, EC 2.4.1.10) as compared to Gram-positive bacteria such as Bacillus subtilis levansucrase. Ea is an enterobacterial pathogen responsible for the Fire Blight disease in rosaceous plants (e.g., apple and pear) with considerable interest for the agricultural industry. The crystal structure of Ea Lsc was solved at 2.77 Å resolution and compared to those of other fructosyltransferases from Gram-positive and Gram-negative bacteria. We propose the structural features, determining the different reaction products, to reside in just a few loops at the rim of the active site funnel. Moreover we propose that loop 8 may have a role in product length determination in Gluconacetobacter diazotrophicus LsdA and Microbacterium saccharophilum FFase. The Ea Lsc structure shows for the first time the products of sucrose hydrolysis still bound in the active site., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A novel method to synthesize highly crosslinked bacterial cellulose (BC) is reported. The glyoxalization is started in-situ, in the culture medium during biosynthesis of cellulose by Gluconacetobacter medellensis bacteria. Strong crosslinked networks were formed in the contact areas between extruded cellulose ribbons by reaction with the glyoxal precursors. The crystalline structure of cellulose was preserved while the acidic component of the surface energy was reduced. As a consequence, its predominant acidic character and the relative contribution of the dispersive component increased, endowing the BC network with a higher hydrophobicity. This route for in-situ crosslinking is expected to facilitate other modifications upon biosynthesis of cellulose ribbons by microorganisms and to engineer the strength and surface energy of their networks., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This work investigates the feasibility of the use of irreversible electroporation (IRE) in the biofabrication of 3D cellulose nanofibril networks via the bacterial strain Gluconacetobacter xylinus. IRE uses electrical pulses to increase membrane permeability by altering the transmembrane potential; past a threshold, damage to the cell becomes too great and leads to cell death. We hypothesized that using IRE to kill the bacteria at specific locations and particular times, we could introduce conduits in the overall scaffold by preventing cellulose biosynthesis locally. Through mathematical modeling and experimental techniques, electrical effects were investigated and the parameters for IRE of G. xylinus were determined. We found that for a specific set of parameters, an applied electric field of 8 to 12.5 kV/cm, producing a local field of 3 kV/cm, was sufficient to kill most of the bacteria and create a localized pore. However, an applied electric field of 17.5 kV/cm was required to kill all. Results suggest that IRE may be an effective tool to create scaffolds with appropriate porosity for orthopedic applications. Ideally, these engineered scaffolds could be used to successfully treat osteochondral defects.

Cellulose producing bacteria were isolated from fruit samples and kombucha tea (a fermented beverage) using CuSO4 solution in modified Watanabe and Yamanaka medium to inhibit yeasts and molds. Six bacterial strains showing cellulose production were isolated and identified by 16S rRNA gene sequencing as Gluconacetobacter xylinus strain DFBT, Ga. xylinus strain dfr-1, Gluconobacter oxydans strain dfr-2, G. oxydans strain dfr-3, Acetobacter orientalis strain dfr-4, and Gluconacetobacter intermedius strain dfr-5. All the cellulose-producing bacteria were checked for the cellulose yield. A potent cellulose-producing bacterium, i.e., Ga. xylinus strain DFBT based on yield (cellulose yield 5.6 g/L) was selected for further studies. Cellulose was also produced in non- conventional media such as pineapple juice medium and hydrolysed corn starch medium. A very high yield of 9.1 g/L cellulose was obtained in pineapple juice medium. Fourier transform infrared spectrometer (FT-IR) analysis of the bacterial cellulose showed the characteristic peaks. Soft cellulose with a very high water holding capacity was produced using limited aeration. Scanning electron microscopy (SEM) was used to analyze the surface characteristics of normal bacterial cellulose and soft cellulose. The structural analysis of the polymer was performed using (13)C solid-state nuclear magnetic resonance (NMR). More interfibrillar space was observed in the case of soft cellulose as compared to normal cellulose. This soft cellulose can find potential applications in the food industry as it can be swallowed easily without chewing.

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.

TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

Bacterial cellulose (BC) synthesized by Gluconacetobacter sucrofermentans CECT 7291 seems to be a good option for the restoration of degraded paper. In this work BC layers are cultivated and purified by two different methods: an alkaline treatment when the culture media contains ethanol and a thermal treatment if the media is free from ethanol. The main goal of these tests was the characterization of BC layers measured in terms of tear and burst indexes, optical properties, SEM, X-ray diffraction, FTIR, degree of polymerization, static and dynamic contact angles, and mercury intrusion porosimetry. The BC layers were also evaluated in the same terms after an aging treatment. Results showed that BC has got high crystallinity index, low internal porosity, good mechanical properties and high stability over time, especially when purified by the alkaline treatment. These features make BC an adequate candidate for degraded paper reinforcement., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This study characterized the cellulosic and non-cellulosic exopolysaccharides (EPS) produced by four Gluconacetobacter strains. The yields of bacterial cellulose and water-soluble polysaccharides were dependent on both carbon source and Gluconacetobacter strain. The carbon substrate also affected the composition of the free EPS. When galactose served as an exclusive carbon source, Gluconacetobacter xylinus (G. xylinus) ATCC 53524 and ATCC 700178 produced a distinct alkaline stable crystalline product, which influenced the crystallization of cellulose. Gluconacetobacter hansenii (G. hansenii) ATCC 23769 and ATCC 53582, however, did not exhibit any significant change in cellulose crystal properties when galactose was used as the carbon source. Microscopic observation further confirmed significant incorporation of EPS into the cellulose composites. The cellulosic network produced from galactose medium showed distinctive morphological and structural features compared to that from glucose medium., (Copyright © 2014 Elsevier Ltd. All rights reserved.)