Characterization of acetic acid bacteria in traditional acetic acid fermentation of rice vinegar (komesu) and unpolished rice vinegar (kurosu) produced in Japan

Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.     

Accumulation of (2,4-dichlorophenoxy) acetic acid and (2,4,5-trichlorophenoxy) acetic acid by Parenchyma Tissue as Influenced by Metabolic Inhibitors and Lecithin

Parenchyma tissue from potato (Solanum tuberosum L. cv. Russet) tubers was treated with inhibitors to the release of metabolic energy in order to determine the importance of an active transport system for (2,4-dichlorophenoxy)acetic acid (2,4-D) and (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T) accumulation. Results from treatments with carbonylcyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation, and N,N'-dicyclohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphosphatase, indicated 2,4-D and 2,4,5-T accumulation to be independent of available energy as influenced by these metabolic inhibitors. Lecithin treated parenchyma tissue accumulated more 2,4-D and 2,4,5-T than untreated tissue indicating possible binding of the herbicide to the lecithin moiety.     

The effect of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae

Summary The effects of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. Cells grown in the presence of acetic acid at a defined specific growth rate showed a higher acetic acid tolerance and a slightly lower trehalose content. Cells grown at a low specific growth rate showed a lower energy demand, a higher acetic acid tolerance, and a higher trehalose content. These results indicate that trehalose plays a growth rate dependent role in the tolerance of S. cerevisiae to acetic acid.     

Site-specific incorporation of (aminooxy)acetic acid into proteins

By employing a general biosynthetic method for the elaboration of proteins containing unnatural amino acid analogues, we incorporated (aminooxy)acetic acid into positions 10 and 27 of Escherichia coli dihydrofolate reductase. Introduction of the modified amino acid into DHFR was accomplished in an in vitro protein biosynthesizing system by readthrough of a nonsense (UAG) codon with a suppressor tRNA that had been activated with (aminooxy)acetic acid. Incorporation of the amino acid proceeded with reasonable efficiency at codon position 10 but less well at position 27. (Aminooxy)acetic acid was also incorporated into position 72 of DNA polymerase beta. Peptides containing (aminooxy)acetic acid have been shown to adopt a preferred conformation involving an eight-membered ring that resembles a gamma-turn. Accordingly, the present study may facilitate the elaboration of proteins containing conformationally biased peptidomimetic motifs at predetermined sites. The present results further extend the examples of ribosomally mediated formation of peptide bond analogues of altered connectivity and provide a conformationally biased linkage at a predetermined site. It has also been shown that the elaborated protein can be cleaved chemically at the site containing the modified amino acid.     

Copper(II) complexes derived from (2-carboxymethoxy-phenylamino)acetic acid and analogue

The synthesis and characterization of mononuclear copper complex of (3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-acetic acid (1) and a tetranuclear copper complex of (2-carboxymethoxy-phenylamino)acetic acid (2) is reported. The sodium salt 1 on reaction with copper(II) chloride hexahydrate followed by treatment with pyridine gave a mononuclear copper complex; whereas, a tetra-nuclear complex in the case of reaction of 2 with copper(II) chloride hexahydrate and 2,2′-bipyridine was obtained. In tetra-nuclear copper(II) complex the NH group co-ordinates to copper and cluster has five co-ordination around copper(II).     

Inhibition of C4 photosynthesis by (benzamidooxy)acetic acid

(Benzamidooxy)acetic acid (common name benzadox) which has herbicidal properties was evaluated as a potential inhibitor of photosynthesis in C₄ plants. Among enzymes of the C₄ pathway, it was a relatively strong inhibitor of alanine aminotransferase in in vitro experiments at concentrations of 5mM. In benzadox treated leaves of Panicum miliaceum, a NAD-malic enzyme type C₄ species, there was strong inhibition of both alanine and aspartate aminotransferase and of photosynthetic O₂ evolution within one hour. Consistent with the inhibition of these enzymes of the C₄ cycle, the pool sizes of metabolites of the cycle was altered: the aspartate level was increased two fold, while the levels of other metabolites such as pyruvate, alanine, oxalacetate and malate were decreased. Kinetic studies with partially purified alanine aminotransferase showed that benzadox is a competitive inhibitor with respect to alanine and a noncompetitive inhibitor with respect to 2-oxoglutarate. Comparisons between the structures and inhibitory actions of benzadox and (aminooxy)acetic acid, the latter a potent inhibitor of alanine and aspartate aminotransferases, suggest that in vivo, benzadox may exert its effect through metabolism to (aminooxy)acetic acid.Abbreviations benzadox (benzamidooxy)acetic acid - DTE dithioerythritol This research was supported in part by gift funds from Monsanto Agricultural Products Company. St. Louis, Missouri, and by NSF Grant PCM-8107953.     

Colorimetric assay of 2,2-bis(4-chlorophenyl)acetic acid (DDA) in urine

2,2-Bis(4-chlorophenyl) acetic acid (DDA) was extracted from acidified urine at pH 2 by benzene. It was complexed by partitioning with crystal violet in acetic acid to yield a blue color which was quantitated at 610 nm. Its identity was confirmed by paper chromatographic separation. The DDA excretion in nine subjects ranged from 0.024 to 0.130 μg/ml with a mean of 0.065 while that determined by gas chromatography ranged from 0.038 to 0.120 with a mean of 0.069.     

Epidermal growth factor (EGF) inhibits both intrinsic factor secretion and acid secretion in histamine-stimulated isolated gastric glands

Epidermal growth factor (EGF) is a polypeptide present in mammalian salivary glands which has been shown to have mitogenic and gastric acid inhibitory properties in vivo. The mechanisms of action of EGF at the level of the parietal cell are not clear. In the present study, we have examined the effects of EGF on both acid and macromolecular (intrinsic factor, IF) secretion stimulated by the cyclic AMP-mediated agonist histamine using the rabbit isolated gastric gland model. Acid secretion was assessed by the accumulation of [14C]aminopyrine (AP) in glands and IF in the supernatants by the binding of [57Co]cyanocobalamin. Histamine (10(-6) to 5 x 10(-5) M) resulted in a 4-6 fold increase in [14C]AP and IF (P less than 0.05). EGF alone (10(-8) M, 10(-7) M) had no significant effect on basal [14C]AP accumulation or IF secretion (P less than 0.05). EGF (10(-7) M) significantly inhibited the histamine dose-response curve for [14C]AP and IF, but a relatively greater inhibition was observed at higher histamine concentration. These data demonstrate that EGF inhibits both acid and IF secretion in vitro at concentrations consistent with those observed in vivo. The observations further support the hypothesis that EGF may play a role in the regulation of parietal cell secretion.     

Conjugated linoleic acid (CLA) inhibits fatty acid synthetase activity in vitro

This paper describes the in vitro effect of conjugated linoleic acid (CLA) on fatty acid biosynthesis. Among the rat liver enzymes involved in fatty acid biosynthesis, fatty acid synthetase (FAS) showed the largest activity fluctuation with the types of fatty acids. Of the fatty acids, CLA was the most potent inhibitor of FAS, and the 9c, 11t-rather than the 10t, 12c-isomer showed greater inhibition. CLA also significantly lowered the incorporation of [14C]-acetate into phospholipid in breast cancer cells, supporting the view that CLA inhibits fatty acid biosynthesis through the interaction with FAS.     

Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria

In this study, we compared the growth properties and molecular characteristics of pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) among highly acetic acid-resistant strains of acetic acid bacteria. Ga. europaeus exhibited the highest resistance to acetic acid (10%), whereas Ga. intermedius and Acetobacter pasteurianus resisted up to 6% of acetic acid. In media with different concentrations of acetic acid, the maximal acetic acid production rate of Ga. europaeus slowly increased, but specific growth rates decreased concomitant with increased concentration of acetic acid in medium. The lag phase of A. pasteurianus was twice and four times longer in comparison to the lag phases of Ga. europaeus and Ga. intermedius, respectively. PQQ-dependent ADH activity was twice as high in Ga. europaeus and Ga. intermedius as in A. pasteurinus. The purified enzymes showed almost the same specific activity to each other, but in the presence of acetic acid, the enzyme activity decreased faster in A. pasteurianus and Ga. intermedius than in Ga. europaeus. These results suggest that high ADH activity in the Ga. europaeus cells and high acetic acid stability of the purified enzyme represent two of the unique features that enable this species to grow and stay metabolically active at extremely high concentrations of acetic acid.     

The effect of the herbicide 2,4,5 trichlorophenoxy acetic acid (245T) on the growth and metabolism of Tetrahymena pyriformis

The herbicide 2,4,5 trichlorophenoxy acetic acid (245T) at concentrations from 0.5 to 0.9 mM, was found to inhibit respiration and then growth in exponentially growing cultures of Tetrahymena pyriformis. Cell division was stopped for periods up to 60 minutes after which the cells recovered and division resumed. Recovery of oxygen utilization and cell division occurred in the presence of 245T. 245T was shown to inhibit mitochondrial oxygen utilization. Mitochondria from cells that had recovered from 245T treatment lost their sensitivity to low concentrations of the herbicide and sedimented deeper in a sucrose gradient than mitochondria from control cells.     

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance

Transforming growth factor-beta 2 (TGF-β2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-β2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-β2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-β2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-β2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm’s canal (MSC) cell monolayers was decreased by TGF-β2 treatment. SAHA inhibited the effects of TGF-β2 on the permeability of these cells. TGF-β2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-β2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-β2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-β signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-β. These results imply that SAHA prevents TGF-β2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-β signaling in TM and MSC cells.     

Effects of acetic acid on growth and fermentative activity of Saccharomyces cerevisiae

The effect of acetic acid on the growth and the fermentative activity of S. cerevisiae was analysed comparatively with the pH. This study showed that the pH does not affect these two activities. On the contrary, the acetic acid has an inhibition effect. This effect was modelised by the relation of Levenspiel. Finally, it was shown that the quantities of acetic acid produced by Brettanomyces were not sufficient to explain the inhibition of Saccharomyces.     

Monitoring growth and acetic acid secretion by a thermotolerantBacillus using conduction microcalorimetry

Summary A method is described to determine power of heat-time curves by conduction microcalorimetry in order to monitor the viability and ability of a thermotolerantBacillus strain to secrete acetic acid both during exponential growth and during stationary-phase. In this system secreted acetic acid is neutralized by an insoluble source of lime (dolime) which results in a poor correlation between optical density and culture dry weight. As an alternative, cells and residual dolime were rapidly resuspended in isothermal fresh medium with glucose in a conduction microcalorimeter. Heat evolution was rapid over a period of 200–800 s. Steady state heat evolution rate decreased as a function of culture time and did not correlate with: 1) specific growth rate: 2) viable cell number: 3) glucose consumption rate; or 4) acetic acid secretion rate. Glucose consumption and acetic acid secretion during the stationary growth phase were correlated with specific heat evolution rate. These initial results indicate that this technique may be useful for further development as an on-line flow or stopped-flow method to monitor the physiology of bacilli in response to nutrient depletion or growth inhibition.     

Effect of yeast extract on Escherichia coli growth and acetic acid production

Fed batch cultures were performed to investigate the effect of yeast extract concentration on the kinetics of growth and acetic acid production of recombinant Escherichia coli BL21 in a synthetic medium. Three runs were performed with 40g/l total glucose concentration. The yeast extract/glucose ratio (YE/G; w/w), was 0.1, 0.05 and 0.025 in the feed. These decreasing YE/G values did not affect growth kinetics, but reduced the final cell concentration by about 10%, and also reduced the cell yield. Experiments with 60g/l total glucose concentration, one with a YE/G of 0.025 in the feed and the other without yeast extract, showed final acetic acid concentrations of 5.1 and 0.5g/l respectively, without any difference in cellular concentration. Although there was no significant influence on growth kinetics and final cellular concentration, the cell fermentative capacity was enhanced by yeast extract. The feed medium without yeast extract was the best condition for control purposes in high cell density cultures and for recombinant gene expression.     

Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium

Specific growth rates (μ) of two strains of Saccharomyces cerevisiae decreased exponentially (R ²>0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30°C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05–0.1% w/v and lactic acid at concentrations of 0.2–0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P≤0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic. Journal of Industrial Microbiology & Biotechnology (2001) 26, 171–177. Received 06 June 2000/ Accepted in revised form 21 September 2000     

Influence of produced acetic acid on growth of sulfate reducing bacteria

Summary A culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7.0. Highest growth rates were observed at pH 6.6. Under gás (H₂S) stripping conditions the specific growth rate decreased with the undissociated acetic acid produced. An inhibition of SRB growth of 50% was observed for undissociated acetic acid concentrations of approximately 54 mg/L.     

Isolation barriers between petunia axillaris and Petunia integrifolia (Solanaceae)

The isolation barriers restricting gene flow between populations or species are of crucial interest for understanding how biological species arise and how they are maintained. Few studies have examined the entire range of possible isolation barriers from geographic isolation to next generation hybrid viability. Here, we present a detailed analysis of isolation barriers between two flowering plant species of the genus Petunia (Solanaceae). Petunia integrifolia and P. axillaris feature divergent pollination syndromes but can produce fertile hybrids when crossed in the laboratory. Both Petunia species are primarily isolated in space but appear not to hybridize in sympatry. Our experiments demonstrate that pollinator isolation is very high but not strong enough to explain the absence of hybrids in nature. However, pollinator isolation in conjunction with male gametic isolation (i.e., pollen-pistil interaction) can explain the lack of natural hybridization, while postzygotic isolation barriers are low or nonexistent. Our study supports the notion that reproductive isolation in flowering plants is mainly caused by pre- rather than postzygotic isolation mechanisms.