Local Necrotic Cells Trigger Systemic Immune Activation via Gut Microbiome Dysbiosis in Drosophila.

Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. Here, we demonstrate that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome.

Introducing a thermotolerant Gluconobacter japonicus strain, potentially useful for coenzyme Q10 production.

In this report, Gluconobacter strains were screened for coenzyme Q10 (CoQ10) production. A thermotolerant strain, Gluconobacter japonicus FM10, was eventually employed for CoQ10 production optimization. To do so, a two-step optimization strategy was used. The first step focused on biomass increase and the second step focused on increase in CoQ10 production. Factors including temperature, pH, carbon, and nitrogen sources were optimized at the first step, and temperature, pH, and aeration were optimized at the second step. The batch culture fermentation was used with the optimized factors of the first phase (30 °C, pH 6.5, D-sorbitol, and yeast extract-peptone as the carbon and nitrogen sources). After 18 h, the temperature, pH, and aeration were shifted to the optimized values of the second step (36 °C, pH 7, and no aeration). By this strategy, the dry cell mass (17.1 g/L) and CoQ10 (23.2 mg/L) were obtained after 20 h, which the latter was 2.3 times higher than that of the first step of optimization. Among the conditions tested, carbon source was the most important factor on the cell growth at the first step while no aeration was the key factor for CoQ10 production in the second step of optimization.

Influence of levan-producing acetic acid bacteria on buckwheat-sourdough breads.

Buckwheat sourdoughs supplemented with molasses as natural sucrose source were fermented with levan-producing Gluconobacter (G.) albidus TMW 2.1191 and Kozakia (K.) baliensis NBRC 16680. Cell growth, concomitant levan and low-molecular-weight metabolite production were monitored. Sourdough breads were prepared with different sourdoughs from both strains (24, 30 and 48 h fermentation, respectively) and analyzed with respect to bread volume, crumb hardness and sensory characteristics. During fermentation, levan, acetic and gluconic acids were increasingly produced, while spontaneously co-growing lactic acid bacteria additionally formed acetic and lactic acids. Sourdoughs from both strains obtained upon 24 h of fermentation significantly improved the bread sensory and quality, including higher specific volume as well as lower crumb hardness. Buckwheat doughs containing isolated levan, with similar molecular size and mass compared to in situ produced levan in the sourdough at 48 h, verified the positive effect of levan on bread quality. However, the positive effects of levan were masked to a certain extent by the impact from the natural acidification during fermentations. While levan-producing acetic acid bacteria are a promising alternative for the development of clean-label gluten-free breads without the need of additives, an appropriate balance between acidification and levan production (amount and structure) must be reached.

Effect of ammonium and amino acids on the growth of selected strains of Gluconobacter and Acetobacter.

Acetic acid bacteria (AAB) are a group of microorganisms highly used in the food industry. However, its use can be limited by the insufficient information known about the nutritional requirements of AAB for optimal growth. The aim of this work was to study the effects of different concentrations and sources of nitrogen on the growth of selected AAB strains and to establish which nitrogen source best encouraged their growth. Two strains of three species of AAB, Gluconobacter japonicus, Gluconobacter oxydans and Acetobacter malorum, were grown in three different media with diverse nitrogen concentrations (25, 50, 100, and 300mgN/L and 1gN/L) as a complete solution of amino acids and ammonium. With this experiment, the most favourable medium and the lowest nitrogen concentration beneficial for the growth of each strain was selected. Subsequently, under these conditions, single amino acids or ammonium were added to media individually to determine the best nitrogen sources for each AAB strain. The results showed that nitrogen requirements are highly dependent on the nitrogen source, the medium and the AAB strain. Gluconobacter strains were able to grow in the lowest nitrogen concentration tested (25mgN/L); however, one of the G. oxydans strains and both A. malorum strains required a higher concentration of nitrogen (100-300mgN/L) for optimal growth. In general, single nitrogen sources were not able to support the growth of these AAB strains as well as the complete solution of amino acids and ammonium.

Chemical mutagenesis of Gluconobacter frateurii to construct methanol-resistant mutants showing glyceric acid production from methanol-containing glycerol.

To produce glyceric acid (GA) from methanol-containing glycerol, resistance to methanol of Gluconobacter frateurii NBRC103465 was improved by chemical mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The obtained mutant Gf398 produced 6.3 g/L GA in 5% (v/v) methanol-containing 17% (w/v) glycerol medium, in which the wild-type strain neither grew nor produced GA.

Expression and characterization of a class III alcohol dehydrogenase gene from Gluconobacter frateurii in the presence of methanol during glyceric acid production from glycerol.

Some acetic acid bacteria have been shown to produce large amounts of glyceric acid (GA) from glycerol, which is a by-product of biodiesel fuel (BDF) production. Previously, a Gluconobacter strain was found that produced decreased amounts of GA from glycerol in the presence of methanol, a major ingredient of raw glycerol derived from the BDF industry. Thus, a comparative transcriptome analysis of Gluconobacter frateurii NBRC103465 was performed to investigate changes in gene expression during GA production from glycerol in the presence of methanol. Cells grown with methanol showed upregulated expression of a class III alcohol dehydrogenase homolog (adhC(Gf)) and decreased GA production. adhC(Gf) was cloned and expressed heterologously in Escherichia coli, and the presence of an additional protein with an approximate molecular mass of 39 kDa in the cytosol of the recombinant E. coli cells was identified by SDS-PAGE. Activity measurements of the cytosol revealed that the translational product of adhC(Gf) exhibited formaldehyde dehydrogenase activity in the presence of nicotinamide adenine dinucleotide and glutathione. Gluconobacter frateurii cells grown in 1% methanol-containing glycerol were found to have fivefold higher formaldehyde dehydrogenase activity than cells grown without methanol, suggesting that adhC(Gf) in G. frateurii cells functions in the dissimilation of methanol-derived formaldehyde.

Conductive photoswitchable vanadium oxide nanopaper based on bacterial cellulose.

A bacterial cellulose mat was used as a template for the fabrication of conductive photoswitchable hybrid nanopaper by the incorporation of sol-gel synthesized vanadium nanoparticles. The resulting nanopaper, prepared through a green pathway, was able to photoinduce a reversible color change. Conductive properties at the nano- and macroscales were confirmed by electrostatic force microscopy and semiconductor analysis measurements, respectively.

High-temperature sorbose fermentation with thermotolerant Gluconobacter frateurii CHM43 and its mutant strain adapted to higher temperature.

We succeeded in obtaining a strain adapted to higher temperature from a thermotolerant strain, Gluconobacter frateurii CHM43, for sorbose fermentation. The adapted strain showed higher growth and L-sorbose production than original CHM43 strain at higher temperature around 38.5-40 °C. It was also shown to be useful even with the fermentation without temperature control. To understand the sorbose fermentation ability of the adapted strain at higher temperature, D-sorbitol-oxidizing respiratory chain was compared with the CHM43 strain and the adapted strain. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which is a primary dehydrogenase of the respiratory chain and responsible for L-sorbose production, was decreased when the temperature increased, but the decreased activity of GLDH was recovered by the addition of PQQ. Since the adapted strain was found to produce more PQQ than the CHM43 strain, it was suggested that the adapted strain keeps GLDH as holoenzyme with the increased PQQ production, and thus produces more L-sorbose and grows better under higher temperature.

Use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol.

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.

Cold atmospheric plasma disinfection of cut fruit surfaces contaminated with migrating microorganisms.

The efficacy of cold atmospheric gas plasmas against Escherichia coli type 1, Saccharomyces cerevisiae, Gluconobacter liquefaciens, and Listeria monocytogenes Scott A was examined on inoculated membrane filters and inoculated fruit surfaces. Inoculated samples were exposed to a cold atmospheric plasma plume generated by an AC voltage of 8 kV at 30 kHz. The cold atmospheric plasma used in this study was very efficient in reducing the microbial load on the surfaces of filter membranes. However, its efficacy was markedly reduced for microorganisms on the cut surfaces. This lack of effect was not the result of quenching of reactive plasma species responsible for microbial inactivation but principally the result of the migration of microorganisms from the exterior of the fruit tissue to its interior. The velocity of migration through melon tissues was estimated to be around 300 microm min(-1) for E. coli and S. cerevisiae and through mango tissues to be 75 to 150 microm min(-1). These data can serve as operational targets for optimizing the performance of gas plasma inactivation processes. The current capabilities of cold atmospheric plasmas are reviewed and ways to improve their bactericidal efficacy are identified and discussed. Considerable scope exists to enhance significantly the efficacy of cold atmospheric plasmas for decontaminating fresh cut fruits.

Energy metabolism of a unique acetic acid bacterium, Asaia bogorensis, that lacks ethanol oxidation activity.

Acetic acid bacteria (AAB) are known as a vinegar producer on account of their ability to accumulate a high concentration of acetic acid due to oxidative fermentation linking the ethanol oxidation respiratory chain. Reactions in oxidative fermentation cause poor growth because a large amount of the carbon source is oxidized incompletely and the harmful oxidized products are accumulated almost stoichiometrically in the culture medium during growth, but a newly identified AAB, Asaia, has shown unusual properties, including scanty acetic acid production and rapid growth, as compared with known AAB as Acetobacter, Gluconobacter, and Gluconacetobacter. To understand these unique properties of Asaia in more detail, the respiratory chain and energetics of this strain were investigated. It was found that Asaia lacks quinoprotein alcohol dehydrogenase, but has other sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter, especially quinoprotein glycerol dehydrogenase. It was also found that Asaia has a cyanide-sensitive cytochrome bo(3)-type ubiquinol oxidase as sole terminal oxidase in the respiratory chain, and that it exhibits a higher H(+)/O ratio.

Distinct physiological roles of two membrane-bound dehydrogenases responsible for D-sorbitol oxidation in Gluconobacter frateurii.

Two different membrane-bound enzymes oxidizing D-sorbitol are found in Gluconobacter frateurii THD32: pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) and FAD-dependent D-sorbitol dehydrogenase (FAD-SLDH). In this study, FAD-SLDH appeared to be induced by L-sorbose. A mutant defective in both enzymes grew as well as the wild-type strain did, indicating that both enzymes are dispensable for growth on D-sorbitol. The strain defective in PQQ-GLDH exhibited delayed L-sorbose production, and lower accumulation of it, corresponding to decreased oxidase activity for D-sorbitol in spite of high D-sorbitol dehydrogenase activity, was observed. In the mutant strain defective in PQQ-GLDH, oxidase activity with D-sorbitol was much more resistant to cyanide, and the H(+)/O ratio was lower than in either the wild-type strain or the mutant strain defective in FAD-SLDH. These results suggest that PQQ-GLDH connects efficiently to cytochrome bo(3) terminal oxidase and that it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO.

Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila.

Although commensalism with gut microbiota exists in all metazoans, the host factors that maintain this homeostatic relationship remain largely unknown. We show that the intestinal homeobox gene Caudal regulates the commensal-gut mutualism by repressing nuclear factor kappa B-dependent antimicrobial peptide genes. Inhibition of Caudal expression in flies via RNA interference led to overexpression of antimicrobial peptides, which in turn altered the commensal population within the intestine. In particular, the dominance of one gut microbe, Gluconobacter sp. strain EW707, eventually led to gut cell apoptosis and host mortality. However, restoration of a healthy microbiota community and normal host survival in the Caudal-RNAi flies was achieved by reintroduction of the Caudal gene. These results reveal that a specific genetic deficiency within a host can profoundly influence the gut commensal microbial community and host physiology.

L-sorbose reductase and its transcriptional regulator involved in L-sorbose utilization of Gluconobacter frateurii.

Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent D-sorbitol dehydrogenase (SLDH), sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent L-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on L-sorbose, indicating that the SboA enzyme is required for efficient growth on L-sorbose. The sboR mutant grew on L-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on D-sorbitol and that another activator is required for the induction of these genes by D-sorbitol or L-sorbose.

Bacterial cellulose production by Gluconacetobacter sp. PKY5 in a rotary biofilm contactor.

A rotary biofilm contactor (RBC) inoculated with Gluconacetobacter sp. RKY5 was used as a bioreactor for improved bacterial cellulose production. The optimal number of disk for bacterial cellulose production was found to be eight, at which bacterial cellulose and cell concentrations were 5.52 and 4.98 g/L. When the aeration rate was maintained at 1.25 vvm, bacterial cellulose and cell concentrations were maximized (5.67 and 5.25 g/L, respectively). The optimal rotation speed of impeller in RBC was 15 rpm. When the culture pH in RBC was not controlled during fermentation, the maximal amount of bacterial cellulose (5.53 g/L) and cells (4.91 g/L) was obtained. Under the optimized culture conditions, bacterial cellulose and cell concentrations in RBC reached to 6.17 and 5.58 g/L, respectively.

Application of molecular methods to demonstrate species and strain evolution of acetic acid bacteria population during wine production.

The growth of acetic acid bacteria on grapes or throughout the winemaking process influences the quality of wine, mainly because it increases the volatile acidity. The objective of this study was to analyse how the acetic acid bacteria population evolves in the changing environment of the grape surface and during wine fermentation. We have analysed the influence of yeast inoculation and SO2 addition on acetic acid bacteria populations. These bacteria were analysed at both the species and the strain level by molecular methods such as Restriction Fragment Length Polimorfism (RFLP) of amplified 16S rDNA, and amplification by polymerase chain reaction of Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) and Repetitive Extragenic Palindromic (REP-PCR). Our results show that the increases in population size are normally accompanied by a proliferation of Acetobacter aceti, which is the main species during fermentation. The diversity of strains is considerable in natural environments such as the grape surface. Changes in the environment during alcoholic fermentation substantially reduce the survival and the diversity of acetic acid bacteria. Few strains are able to survive these conditions and they seem to originate from both the grapes and the winery. To the best of our knowledge this is the first time that acetic acid bacteria are analysed at the strain level in grape surfaces and during winemaking.

Gluconobacter asaii Mason and Claus 1989 is a junior subjective synonym of Gluconobacter cerinus Yamada and Akita 1984.

Five strains received as Gluconobacter cerinus and Gluconobacter asaii were examined for DNA base composition, DNA-DNA similarity, 16S rRNA gene sequences and phenotypic characteristics, including acid production from ethanol, growth on L-arabitol and meso-ribitol and requirement for nicotinic acid. The five strains showed DNA base compositions ranging from 54 to 56 mol% G+C. G. cerinus IFO 3267T and IAM 1832 and G. asaii IFO 3276T and IFO 3275 showed high levels of DNA-DNA similarity (70-100%) between each other and low values of DNA-DNA similarity (16-35%) to Gluconobacter frateurii IFO 3264T and Gluconobacter oxydans IFO 14819T. G. cerinus IFO 3267T and G. asaii IFO 3276T were located at an identical position in a phylogenetic tree deduced from 16S rRNA gene sequences. Two G. cerinus strains and two G. asaii strains did not require nicotinic acid for growth and did not grow on L-arabitol or meso-ribitol. G. cerinus IAM 1832 did not produce acid and required nicotinic acid and/or other growth factors. G. asaii IFO 3265 showed a high degree of DNA-DNA similarity (97%) to G. frateurii IFO 3264T and low similarity values (each 32%) to G. cerinus IFO 3267T and G. asaii IFO 3276T. This strain did not require nicotinic acid and grew well on L-arabitol and meso-ribitol. Therefore, G. asaii IFO 3265 was reclassified as G. frateurii. The results obtained revealed a synonymous relationship between G. cerinus and G. asaii. G. asaii is a junior subjective synonym of G. cerinus because G. cerinus has priority over G. asaii.

Role of botrytized grape micro-organisms in SO2 binding phenomena.

AIMS: The purpose of this work was to study the involvement of micro-organisms, which develop together with Botrytis cinerea on grapes, in the SO2 binding power of musts. METHODS AND RESULTS: Yeasts and bacteria were involved. Most bacteria were acetic acid bacteria, mainly of the Gluconobacter genus. Unlike oxidative yeasts, Gluconobacter produce gluconic acid (in balance with delta-gluconolactone) from glucose, 5-oxofructose from fructose and dihydroxyacetone from glycerol. Production of carbonyl compounds from other sugars and polyols was not detected or was very weak. CONCLUSION: Acetic acid bacteria are responsible for the increases in SO2 binding power of musts from botrytized grapes by oxidizing the three main sugars of these grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: Up to 80% of the SO2 binds with products of Gluconobacter which easily grow on 'botrytized' grapes. Depending on climatic conditions, some vintages are particularly difficult to stabilize.

Effect of glycine betaine on osmoadaptation of Propionibacterium acidipropionici cultivated in elevated osmolarities.

The sensitivity of industrial strains Acetobacter aceti, Gluconobacter frateurii, and Propionibacterium acidipropionici to osmotic stress was studied. Growth of A. aceti and G. frateurii was totally inhibited at 0.4 M NaCl concentration, but P. acidipropionici was able to grow on a medium containing 1.2 M NaCl. Addition of glycine betaine to the medium had no detectable osmoprotective effect on A. aceti and G. frateurii cultivations in elevated NaCl concentrations, but it enabled cells of P. acidipropionici to achieve faster the maximum specific growth rate after the prolonged lag phase and therefore to gain faster the final biomass and product concentrations. The final concentrations of biomass and product of P. acidipropionici were the same as for the cultivations of the bacterium without NaCl and glycine betaine present in the medium. Intracellular accumulation of glycine betaine was detected in P. acidipropionici cells cultivated in the medium containing glycine betaine. The amount accumulated increased with NaCl concentration, suggesting that glycine betaine plays an important role in the osmoadaptation.