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Sample records for actinobacillus succinogenes atcc

  1. Significance of CO2 donor on the production of succinic acid by Actinobacillus succinogenes ATCC 55618

    PubMed Central

    2011-01-01

    Background Succinic acid is a building-block chemical which could be used as the precursor of many industrial products. The dissolved CO2 concentration in the fermentation broth could strongly regulate the metabolic flux of carbon and the activity of phosphoenolpyruvate (PEP) carboxykinase, which are the important committed steps for the biosynthesis of succinic acid by Actinobacillus succinogenes. Previous reports showed that succinic acid production could be promoted by regulating the supply of CO2 donor in the fermentation broth. Therefore, the effects of dissolved CO2 concentration and MgCO3 on the fermentation process should be investigated. In this article, we studied the impacts of gaseous CO2 partial pressure, dissolved CO2 concentration, and the addition amount of MgCO3 on succinic acid production by Actinobacillus succinogenes ATCC 55618. We also demonstrated that gaseous CO2 could be removed when MgCO3 was fully supplied. Results An effective CO2 quantitative mathematical model was developed to calculate the dissolved CO2 concentration in the fermentation broth. The highest succinic acid production of 61.92 g/L was obtained at 159.22 mM dissolved CO2 concentration, which was supplied by 40 g/L MgCO3 at the CO2 partial pressure of 101.33 kPa. When MgCO3 was used as the only CO2 donor, a maximal succinic acid production of 56.1 g/L was obtained, which was just decreased by 7.03% compared with that obtained under the supply of gaseous CO2 and MgCO3. Conclusions Besides the high dissolved CO2 concentration, the excessive addition of MgCO3 was beneficial to promote the succinic acid synthesis. This was the first report investigating the replaceable of gaseous CO2 in the fermentation of succinic acid. The results obtained in this study may be useful for reducing the cost of succinic acid fermentation process. PMID:22040346

  2. Actinobacillus succinogenes ATCC 55618 Fermentation Medium Optimization for the Production of Succinic Acid by Response Surface Methodology

    PubMed Central

    Zhu, Li-Wen; Wang, Cheng-Cheng; Liu, Rui-Sang; Li, Hong-Mei; Wan, Duan-Ji; Tang, Ya-Jie

    2012-01-01

    As a potential intermediary feedstock, succinic acid takes an important place in bulk chemical productions. For the first time, a method combining Plackett-Burman design (PBD), steepest ascent method (SA), and Box-Behnken design (BBD) was developed to optimize Actinobacillus succinogenes ATCC 55618 fermentation medium. First, glucose, yeast extract, and MgCO3 were identified to be key medium components by PBD. Second, preliminary optimization was run by SA method to access the optimal region of the key medium components. Finally, the responses, that is, the production of succinic acid, were optimized simultaneously by using BBD, and the optimal concentration was located to be 84.6 g L−1 of glucose, 14.5 g L−1 of yeast extract, and 64.7 g L−1 of MgCO3. Verification experiment indicated that the maximal succinic acid production of 52.7 ± 0.8 g L−1 was obtained under the identified optimal conditions. The result agreed with the predicted value well. Compared with that of the basic medium, the production of succinic acid and yield of succinic acid against glucose were enhanced by 67.3% and 111.1%, respectively. The results obtained in this study may be useful for the industrial commercial production of succinic acid. PMID:23093852

  3. Development of a Markerless Knockout Method for Actinobacillus succinogenes

    PubMed Central

    Joshi, Rajasi V.; Schindler, Bryan D.; McPherson, Nikolas R.; Tiwari, Kanupriya

    2014-01-01

    Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain. PMID:24610845

  4. Economical succinic acid production from cane molasses by Actinobacillus succinogenes.

    PubMed

    Liu, Yu-Peng; Zheng, Pu; Sun, Zhi-Hao; Ni, Ye; Dong, Jin-Jun; Zhu, Lei-Lei

    2008-04-01

    In this work, production of succinic acid by Actinobacillus succinogenes CGMCC1593 using cane molasses as a low cost carbon source was developed. In anaerobic bottles fermentation, succinic acid concentration of 50.6+/-0.9 g l(-1) was attained at 60 h using an optimum medium containing molasses pretreated with sulfuric acid, resulting in a succinic acid yield of 79.5+/-1.1% and sugar utilization of 97.1+/-0.6%. When batch fermentation was carried out in a 5-l stirred bioreactor with pretreated molasses, 46.4 g l(-1) of succinic acid was attained at 48 h and faster cells growth was also observed. Fed batch fermentation was performed to minimize the substrate (sugar) inhibition effect, giving 55.2 g l(-1) of succinic acid and 1.15 g l(-1)h(-1) of productivity at 48 h. The present study suggests that the inexpensive cane molasses could be utilized for the economical and efficient production of succinic acid by A. succinogenes.

  5. Continuous succinic acid production from xylose by Actinobacillus succinogenes.

    PubMed

    Bradfield, Michael F A; Nicol, Willie

    2016-02-01

    Continuous, anaerobic fermentations of D-xylose were performed by Actinobacillus succinogenes 130Z in a custom, biofilm reactor at dilution rates of 0.05, 0.10 and 0.30 h(-1). Succinic acid yields on xylose (0.55-0.68 g g(-1)), titres (10.9-29.4 g L(-1)) and productivities (1.5-3.4 g L(-1) h(-1)) were lower than those of a previous study on glucose, but product ratios (succinic acid/acetic acid = 3.0-5.0 g g(-1)) and carbohydrate consumption rates were similar. Also, mass balance closures on xylose were up to 18.2 % lower than those on glucose. A modified HPLC method revealed pyruvic acid excretion at appreciable concentrations (1.2-1.9 g L(-1)) which improved the mass balance closure by up to 16.8 %. Furthermore, redox balances based on the accounted xylose consumed and the excreted metabolites, indicated an overproduction of reducing power. The oxidative pentose phosphate pathway was shown to be a plausible source of the additional reducing power. PMID:26610345

  6. Simultaneous saccharification and fermentation of acid-pretreated rapeseed meal for succinic acid production using Actinobacillus succinogenes.

    PubMed

    Chen, Kequan; Zhang, Han; Miao, Yelian; Wei, Ping; Chen, Jieyu

    2011-04-01

    Rapeseed meal was evaluated for succinic acid production by simultaneous saccharification and fermentation using Actinobacillus succinogenes ATCC 55618. Diluted sulfuric acid pretreatment and subsequent hydrolysis with pectinase was used to release sugars from rapeseed meal. The effects of culture pH, pectinase loading and yeast extract concentration on succinic acid production were investigated. When simultaneous saccharification and fermentation of diluted acid pretreated rapeseed meal with a dry matter content of 12.5% (w/v) was performed at pH 6.4 and a pectinase loading of 2% (w/w, on dry matter) without supplementation of yeast extract, a succinic acid concentration of 15.5 g/L was obtained at a yield of 12.4 g/100g dry matter. Fed-batch simultaneous saccharification and fermentation was carried out with supplementation of concentrated pretreated rapeseed meal and pectinase at 18 and 28 h to yield a final dry matter content of 20.5% and pectinase loading of 2%, with the succinic acid concentration enhanced to 23.4 g/L at a yield of 11.5 g/100g dry matter and a productivity of 0.33 g/(Lh). This study suggests that rapeseed meal may be an alternative substrate for the efficient production of succinic acid by A. succinogenes without requiring nitrogen source supplementation.

  7. Production of succinic acid from oil palm empty fruit bunch cellulose using Actinobacillus succinogenes

    NASA Astrophysics Data System (ADS)

    Pasma, Satriani Aga; Daik, Rusli; Maskat, Mohamad Yusof

    2013-11-01

    Succinic acid is a common metabolite in plants, animals and microorganisms. It has been used widely in agricultural, food and pharmaceutical industries. Enzymatic hydrolysate glucose from oil palm empty fruit bunch (OPEFB) cellulose was used as a substrate for succinic acid production using Actinobacillus succinogenes. Using cellulose extraction from OPEFB can enhance the production of glucose as a main substrate for succinic acid production. The highest concentration of glucose produced from enzymatic hydrolysis is 167 mg/mL and the sugar recovery is 0.73 g/g of OPEFB. By optimizing the culture medium for succinic acid fermentation with enzymatic hydrolysate of OPEFB cellulose, the nitrogen sources could be reduced to just only 2.5 g yeast extract and 2.5 g corn step liquor. Batch fermentation was carried out using enzymatic hydrolysate of OPEFB cellulose with yeast extract, corn steep liquor and the salts mixture, 23.5 g/L succinic acid was obtained with consumption of 72 g/L glucose in enzymatic hydrolysate of OPEFB cellulose at 38 hours and 37°C. This study suggests that enzymatic hydrolysate of OPEFB cellulose maybe an alternative substrate for the efficient production of succinic acid by Actinobacillus succinogenes.

  8. Succinic acid production from duckweed (Landoltia punctata) hydrolysate by batch fermentation of Actinobacillus succinogenes GXAS137.

    PubMed

    Shen, Naikun; Wang, Qingyan; Zhu, Jing; Qin, Yan; Liao, Siming; Li, Yi; Zhu, Qixia; Jin, Yanling; Du, Liqin; Huang, Ribo

    2016-07-01

    Duckweed is potentially an ideal succinic acid (SA) feedstock due to its high proportion of starch and low lignin content. Pretreatment methods, substrate content and nitrogen source were investigated to enhance the bioconversion of duckweed to SA and to reduce the costs of production. Results showed that acid hydrolysis was an effective pretreatment method because of its high SA yield. The optimum substrate concentration was 140g/L. The optimum substrate concentration was 140g/L. Corn steep liquor powder could be considered a feasible and inexpensive alternative to yeast extract as a nitrogen source. Approximately 57.85g/L of SA was produced when batch fermentation was conducted in a 1.3L stirred bioreactor. Therefore, inexpensive duckweed can be a promising feedstock for the economical and efficient production of SA through fermentation by Actinobacillus succinogenes GXAS137. PMID:27023386

  9. Succinic acid production from duckweed (Landoltia punctata) hydrolysate by batch fermentation of Actinobacillus succinogenes GXAS137.

    PubMed

    Shen, Naikun; Wang, Qingyan; Zhu, Jing; Qin, Yan; Liao, Siming; Li, Yi; Zhu, Qixia; Jin, Yanling; Du, Liqin; Huang, Ribo

    2016-07-01

    Duckweed is potentially an ideal succinic acid (SA) feedstock due to its high proportion of starch and low lignin content. Pretreatment methods, substrate content and nitrogen source were investigated to enhance the bioconversion of duckweed to SA and to reduce the costs of production. Results showed that acid hydrolysis was an effective pretreatment method because of its high SA yield. The optimum substrate concentration was 140g/L. The optimum substrate concentration was 140g/L. Corn steep liquor powder could be considered a feasible and inexpensive alternative to yeast extract as a nitrogen source. Approximately 57.85g/L of SA was produced when batch fermentation was conducted in a 1.3L stirred bioreactor. Therefore, inexpensive duckweed can be a promising feedstock for the economical and efficient production of SA through fermentation by Actinobacillus succinogenes GXAS137.

  10. Succinic acid production from corn stover by simultaneous saccharification and fermentation using Actinobacillus succinogenes.

    PubMed

    Zheng, Pu; Fang, Lin; Xu, Yan; Dong, Jin-Jun; Ni, Ye; Sun, Zhi-Hao

    2010-10-01

    Simultaneous saccharification and fermentation (SSF) technique was applied for succinic acid production by Actinobacillus succinogenes in a 5-l stirred bioreactor with corn stover as the raw material. The process parameters of SSF, including corn stover pretreatment condition, substrate concentration, enzyme loading and fermentation temperature were investigated. Results indicated that pretreating corn stover with diluted alkaline was beneficial for the succinic acid production, and succinic acid yield could be significantly increased when adding the cellulase supplemented with cellobiase. The maximal succinic acid concentration and yield could reach 47.4 g/l and 0.72 g/g-substrate, respectively. The corresponding operation conditions were summarized as follows: SSF operation at 38 °C for 48 h, diluted alkaline pretreated corn stover as substrate with concentration of 70 g/l, enzyme loading of 20FPU cellulase and 10 U cellobiase per gram substrate. This result suggested an industrial potential of succinic acid production by using SSF and corn stover.

  11. Improving succinic acid production by Actinobacillus succinogenes from raw industrial carob pods.

    PubMed

    Carvalho, Margarida; Roca, Christophe; Reis, Maria A M

    2016-10-01

    Carob pods are an inexpensive by-product of locust bean gum industry that can be used as renewable feedstock for bio-based succinic acid. Here, for the first time, unprocessed raw carob pods were used to extract a highly enriched sugar solution, afterwards used as substrate to produce succinic acid using Actinobacillus succinogenes. Batch fermentations containing 30g/L sugars resulted in a production rate of 1.67gSA/L.h and a yield of 0.39gSA/g sugars. Taking advantage of A. succinogenes' metabolism, uncoupling cell growth from succinic acid production, a fed-batch mode was implemented to increase succinic acid yield and reduce by-products formation. This strategy resulted in a succinic acid yield of 0.94gSA/g sugars, the highest yield reported in the literature for fed-batch and continuous experiments, while maintaining by-products at residual values. Results demonstrate that raw carob pods are a highly efficient feedstock for bio-based succinic acid production.

  12. Succinic Acid Production from Cheese Whey using Actinobacillus succinogenes 130 Z

    NASA Astrophysics Data System (ADS)

    Wan, Caixia; Li, Yebo; Shahbazi, Abolghasem; Xiu, Shuangning

    Actinobacillus succinogenes 130 Z was used to produce succinic acid from cheese whey in this study. At the presence of external CO2 supply, the effects of initial cheese whey concentration, pH, and inoculum size on the succinic acid production were studied. The by-product formation during the fermentation process was also analyzed. The highest succinic acid yield of 0.57 was obtained at initial cheese whey concentration of 50 g/L, while the highest succinic acid productivity of 0.58 g h-1 L-1 was obtained at initial cheese whey concentration of 100 g/L. Increase in pH and inoculum size caused higher succinic acid yield and productivity. At the preferred fermentation condition of pH 6.8, inoculum size of 5% and initial cheese whey concentration of 50 g/L, succinic acid yield of 0.57, and productivity of 0.44 g h-1 L-1 were obtained. Acetic acid and formic acid were the main by-products throughout the fermentation run of 48 h. It is feasible to produce succinic acid using lactose from cheese whey as carbon resource by A. succinogenes 130 Z.

  13. Succinic acid production by Actinobacillus succinogenes using hydrolysates of spent yeast cells and corn fiber.

    PubMed

    Chen, Ke-Quan; Li, Jian; Ma, Jiang-Feng; Jiang, Min; Wei, Ping; Liu, Zhong-Min; Ying, Han-Jie

    2011-01-01

    The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources. PMID:20801644

  14. Improving succinic acid production by Actinobacillus succinogenes from raw industrial carob pods.

    PubMed

    Carvalho, Margarida; Roca, Christophe; Reis, Maria A M

    2016-10-01

    Carob pods are an inexpensive by-product of locust bean gum industry that can be used as renewable feedstock for bio-based succinic acid. Here, for the first time, unprocessed raw carob pods were used to extract a highly enriched sugar solution, afterwards used as substrate to produce succinic acid using Actinobacillus succinogenes. Batch fermentations containing 30g/L sugars resulted in a production rate of 1.67gSA/L.h and a yield of 0.39gSA/g sugars. Taking advantage of A. succinogenes' metabolism, uncoupling cell growth from succinic acid production, a fed-batch mode was implemented to increase succinic acid yield and reduce by-products formation. This strategy resulted in a succinic acid yield of 0.94gSA/g sugars, the highest yield reported in the literature for fed-batch and continuous experiments, while maintaining by-products at residual values. Results demonstrate that raw carob pods are a highly efficient feedstock for bio-based succinic acid production. PMID:27394995

  15. Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

    PubMed

    Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-07-01

    Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production. PMID:26996628

  16. Carob pod water extracts as feedstock for succinic acid production by Actinobacillus succinogenes 130Z.

    PubMed

    Carvalho, Margarida; Roca, Christophe; Reis, Maria A M

    2014-10-01

    Carob pods are a by-product of locust bean gum industry containing more than 50% (w/w) sucrose, glucose and fructose. In this work, carob pod water extracts were used, for the first time, for succinic acid production by Actinobacillus succinogenes 130Z. Kinetic studies of glucose, fructose and sucrose consumption as individual carbon sources till 30g/L showed no inhibition on cell growth, sugar consumption and SA production rates. Sugar extraction from carob pods was optimized varying solid/liquid ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Batch fermentations containing 10-15g/L total sugars resulted in a maximum specific SA production rate of 0.61Cmol/Cmol X.h, with a yield of 0.55Cmol SA/Cmol sugar and a volumetric productivity of 1.61g SA/L.h. Results demonstrate that carob pods can be a promising low cost feedstock for bio-based SA production.

  17. Carob pod water extracts as feedstock for succinic acid production by Actinobacillus succinogenes 130Z.

    PubMed

    Carvalho, Margarida; Roca, Christophe; Reis, Maria A M

    2014-10-01

    Carob pods are a by-product of locust bean gum industry containing more than 50% (w/w) sucrose, glucose and fructose. In this work, carob pod water extracts were used, for the first time, for succinic acid production by Actinobacillus succinogenes 130Z. Kinetic studies of glucose, fructose and sucrose consumption as individual carbon sources till 30g/L showed no inhibition on cell growth, sugar consumption and SA production rates. Sugar extraction from carob pods was optimized varying solid/liquid ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Batch fermentations containing 10-15g/L total sugars resulted in a maximum specific SA production rate of 0.61Cmol/Cmol X.h, with a yield of 0.55Cmol SA/Cmol sugar and a volumetric productivity of 1.61g SA/L.h. Results demonstrate that carob pods can be a promising low cost feedstock for bio-based SA production. PMID:25164341

  18. Succinic acid production by Actinobacillus succinogenes from batch fermentation of mixed sugars.

    PubMed

    Almqvist, Henrik; Pateraki, Chrysanthi; Alexandri, Maria; Koutinas, Apostolis; Lidén, Gunnar

    2016-08-01

    Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8-26.8 g/L and a total yield of 0.59-0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14-0.20 g/g and formate 0.08-0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO2 sparging could replace carbonate supply in the form of MgCO3 without affecting the succinate yield. PMID:27255975

  19. Bagasse hydrolyzates from Agave tequilana as substrates for succinic acid production by Actinobacillus succinogenes in batch and repeated batch reactor.

    PubMed

    Corona-González, Rosa Isela; Varela-Almanza, Karla María; Arriola-Guevara, Enrique; Martínez-Gómez, Álvaro de Jesús; Pelayo-Ortiz, Carlos; Toriz, Guillermo

    2016-04-01

    The aim of this work was to obtain fermentable sugars by enzymatic or acid hydrolyses of Agave tequilana Weber bagasse in order to produce succinic acid with Actinobacillus succinogenes. Hydrolyses were carried out with mineral acids (sulfuric and hydrochloric acids) or a commercial cellulolytic enzyme, and were optimized statistically by a response surface methodology, having as factors the concentration of acid/enzyme and time of hydrolysis. The concentration of sugars obtained at optimal conditions for each hydrolysis were 21.7, 22.4y 19.8g/L for H2SO4, HCl and the enzymatic preparation respectively. Concerning succinic acid production, the enzymatic hydrolyzates resulted in the highest yield (0.446g/g) and productivity (0.57g/Lh) using A. succinogenes in a batch reactor system. Repeated batch fermentation with immobilized A. succinogenes in agar and with the enzymatic hydrolyzates resulted in a maximum concentration of succinic acid of 33.6g/L from 87.2g/L monosaccharides after 5 cycles in 40h, obtaining a productivity of 1.32g/Lh.

  20. Bagasse hydrolyzates from Agave tequilana as substrates for succinic acid production by Actinobacillus succinogenes in batch and repeated batch reactor.

    PubMed

    Corona-González, Rosa Isela; Varela-Almanza, Karla María; Arriola-Guevara, Enrique; Martínez-Gómez, Álvaro de Jesús; Pelayo-Ortiz, Carlos; Toriz, Guillermo

    2016-04-01

    The aim of this work was to obtain fermentable sugars by enzymatic or acid hydrolyses of Agave tequilana Weber bagasse in order to produce succinic acid with Actinobacillus succinogenes. Hydrolyses were carried out with mineral acids (sulfuric and hydrochloric acids) or a commercial cellulolytic enzyme, and were optimized statistically by a response surface methodology, having as factors the concentration of acid/enzyme and time of hydrolysis. The concentration of sugars obtained at optimal conditions for each hydrolysis were 21.7, 22.4y 19.8g/L for H2SO4, HCl and the enzymatic preparation respectively. Concerning succinic acid production, the enzymatic hydrolyzates resulted in the highest yield (0.446g/g) and productivity (0.57g/Lh) using A. succinogenes in a batch reactor system. Repeated batch fermentation with immobilized A. succinogenes in agar and with the enzymatic hydrolyzates resulted in a maximum concentration of succinic acid of 33.6g/L from 87.2g/L monosaccharides after 5 cycles in 40h, obtaining a productivity of 1.32g/Lh. PMID:26802183

  1. Use of corn steep liquor as an economical nitrogen source for biosuccinic acid production by Actinobacillus succinogenes

    NASA Astrophysics Data System (ADS)

    Tan, J. P.; Jahim, J. M.; Wu, T. Y.; Harun, S.; Mumtaz, T.

    2016-06-01

    Expensive raw materials are the driving force that leads to the shifting of the petroleum-based succinic acid production into bio-based succinic acid production by microorganisms. Cost of fermentation medium is among the main factors contributing to the total production cost of bio-succinic acid. After carbon source, nitrogen source is the second largest component of the fermentation medium, the cost of which has been overlooked for the past years. The current study aimed at replacing yeast extract- a costly nitrogen source with corn steep liquor for economical production of bio-succinic acid by Actinobacillus succinogenes 130Z. In this study, a final succinic acid concentration of 20.6 g/L was obtained from the use of corn steep liquor as the nitrogen source, which was comparable with the use of yeast extract as the nitrogen source that had a final succinate concentration of 21.4 g/l. In terms of economical wise, corn steep liquor was priced at 200 /ton, which was one fifth of the cost of yeast extract at 1000 /ton. Therefore, corn steep liquor can be considered as a potential nitrogen source in biochemical industries instead of the costly yeast extract.

  2. Utilization of CO2 fixating bacterium Actinobacillus succinogenes 130Z for simultaneous biogas upgrading and biosuccinic acid production.

    PubMed

    Gunnarsson, Ingólfur B; Alvarado-Morales, Merlin; Angelidaki, Irini

    2014-10-21

    Biogas is an attractive renewable energy carrier. However, it contains CO2 which limits its use for certain applications. Here we report a novel approach for removing CO2 from biogas and capturing it as a biochemical through a biological process. This approach entails converting CO2 into biosuccinic acid using the bacterial strain Actinobacillus succinogenes 130 Z, and simultaneously producing high-purity CH4 (> 95%). Results showed that when pressure during fermentation was increased from 101.325 to 140 kPa, higher CO2 solubility was achieved, thereby positively affecting final succinic acid yield and titer, CO2 consumption rate, and CH4 purity. When using biogas as the only CO2 source at 140 kPa, the CO2 consumption rate corresponded to 2.59 L CO2 L(-1) d(-1) with a final succinic acid titer of 14.4 g L(-1). Under this pressure condition, the highest succinic acid yield and biogas quality reached corresponded to 0.635 g g(-1) and 95.4% (v v(-1)) CH4 content, respectively, after 24 h fermentation. This work represents the first successful attempt to develop a system capable of upgrading biogas to vehicle fuel/gas grid quality and simultaneously produce biosuccinic acid, a valuable building block with large market potential in the near term.

  3. Complete genome sequence of Actinobacillus equuli subspecies equuli ATCC 19392T

    PubMed Central

    2015-01-01

    Actinobacillus equuli subsp. equuli is a member of the family Pasteurellaceae that is a common resident of the oral cavity and alimentary tract of healthy horses. At the same time, it can also cause a fatal septicemia in foals, commonly known as sleepy foal disease or joint ill disease. In addition, A. equuli subsp. equuli has recently been reported to act as a primary pathogen in breeding sows and piglets. To better understand how A. equuli subsp. equuli can cause disease, the genome of the type strain of A. equuli subsp. equuli, ATCC 19392T, was sequenced using the PacBio RSII sequencing system. Its genome is comprised of 2,431,533 bp and is predicted to encode 2,264 proteins and 82 RNAs. PMID:26203343

  4. Cellular fatty acid and soluble protein composition of Actinobacillus actinomycetemcomitans and related organisms.

    PubMed Central

    Calhoon, D A; Mayberry, W R; Slots, J

    1981-01-01

    The cellular fatty acid and protein content of twenty-five representative strains of Actinobacillus actinomycetecomitans isolated from juvenile and adult periodontitis patients was compared to that of 15 reference strains of oral and nonoral Actinobacillus species and Haemophilus aphrophilus. Trimethylsilyl derivatives of the fatty acid methyl esters were analyzed by gas-liquid chromatography. The predominant fatty acids of all 40 strains examined were 14:0, 3-OH 14:0, 16 delta, and 16:0. Actinobacillus seminis (ATCC 15768) was unlike the other strains examined because of a greater amount of 14:0 detected. The soluble protein analysis using polyacrylamide gel electrophoresis revealed that A. actinomycetemcomitans, H. aphrophilus, and nonoral Actinobacillus species possessed distinct protein profiles attesting to the validity of separating these organisms into different species. Established biotypes of A. actinomycetemcomitans could not be differentiated on the basis of fatty acid or protein profiles. PMID:7287893

  5. Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Place, D A; Scidmore, N C; McArthur, W P

    1988-01-01

    Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified. PMID:3356470

  6. Succinic acid production on xylose-enriched biorefinery streams by Actinobacillus succinogenes in batch fermentation

    DOE PAGES

    Salvachua, Davinia; Mohagheghi, Ali; Smith, Holly; Bradfield, Michael F. A.; Nicol, Willie; Black, Brenna A.; Biddy, Mary J.; Dowe, Nancy; Beckham, Gregg T.

    2016-02-02

    Co-production of chemicals from lignocellulosic biomass alongside fuels holds promise for improving the economic outlook of integrated biorefineries. In current biochemical conversion processes that use thermochemical pretreatment and enzymatic hydrolysis, fractionation of hemicellulose-derived and cellulose-derived sugar streams is possible using hydrothermal or dilute acid pretreatment (DAP), which then offers a route to parallel trains for fuel and chemical production from xylose- and glucose-enriched streams. Succinic acid (SA) is a co-product of particular interest in biorefineries because it could potentially displace petroleum-derived chemicals and polymer precursors for myriad applications. Furthermore, SA production from biomass-derived hydrolysates has not yet been fully exploredmore » or developed.« less

  7. Continuous Succinic Acid Production by Actinobacillus succinogenes on Xylose-Enriched Hydrolysate

    DOE PAGES

    Bradfield, Michael F. A.; Mohagheghi, Ali; Salvachua, Davinia; Smith, Holly; Black, Brenna A.; Dowe, Nancy; Beckham, Gregg T.; Nicol, Willie

    2015-11-14

    Bio-manufacturing of high-value chemicals in parallel to renewable biofuels has the potential to dramatically improve the overall economic landscape of integrated lignocellulosic biorefineries. However, this will require the generation of carbohydrate streams from lignocellulose in a form suitable for efficient microbial conversion and downstream processing appropriate to the desired end use, making overall process development, along with selection of appropriate target molecules, crucial to the integrated biorefinery. Succinic acid (SA), a high-value target molecule, can be biologically produced from sugars and has the potential to serve as a platform chemical for various chemical and polymer applications. However, the feasibility ofmore » microbial SA production at industrially relevant productivities and yields from lignocellulosic biorefinery streams has not yet been reported.« less

  8. Continuous Succinic Acid Production by Actinobacillus succinogenes on Xylose-Enriched Hydrolysate

    SciTech Connect

    Bradfield, Michael F. A.; Mohagheghi, Ali; Salvachua, Davinia; Smith, Holly; Black, Brenna A.; Dowe, Nancy; Beckham, Gregg T.; Nicol, Willie

    2015-11-14

    Bio-manufacturing of high-value chemicals in parallel to renewable biofuels has the potential to dramatically improve the overall economic landscape of integrated lignocellulosic biorefineries. However, this will require the generation of carbohydrate streams from lignocellulose in a form suitable for efficient microbial conversion and downstream processing appropriate to the desired end use, making overall process development, along with selection of appropriate target molecules, crucial to the integrated biorefinery. Succinic acid (SA), a high-value target molecule, can be biologically produced from sugars and has the potential to serve as a platform chemical for various chemical and polymer applications. However, the feasibility of microbial SA production at industrially relevant productivities and yields from lignocellulosic biorefinery streams has not yet been reported.

  9. Ultrasonic pretreatment and acid hydrolysis of sugarcane bagasse for succinic acid production using Actinobacillus succinogenes.

    PubMed

    Xi, Yong-lan; Dai, Wen-yu; Xu, Rong; Zhang, Jiu-hua; Chen, Ke-quan; Jiang, Min; Wei, Ping; Ouyang, Ping-kai

    2013-11-01

    Immense interest has been devoted to the production of bulk chemicals from lignocellulose biomass. Diluted sulfuric acid treatment is currently one of the main pretreatment methods. However, the low total sugar concentration obtained via such pretreatment limits industrial fermentation systems that use lignocellulosic hydrolysate. Sugarcane bagasse hemicellulose hydrolysate is used as the carbon and nitrogen sources to achieve a green and economical production of succinic acid in this study. Sugarcane bagasse was ultrasonically pretreated for 40 min, with 43.9 g/L total sugar obtained after dilute acid hydrolysis. The total sugar concentration increased by 29.5 %. In a 3-L fermentor, using 30 g/L non-detoxified total sugar as the carbon source, succinic acid production increased to 23.7 g/L with a succinic acid yield of 79.0 % and a productivity of 0.99 g/L/h, and 60 % yeast extract in the medium could be reduced. Compared with the detoxified sugar preparation method, succinic acid production and yield were improved by 20.9 and 20.2 %, respectively. PMID:23649828

  10. An unconventional anaerobic membrane protein production system based on Wolinella succinogenes.

    PubMed

    Lafontaine, Michael; Lancaster, C Roy D

    2015-01-01

    In cases where membrane protein production attempts in more conventional Escherichia coli-based systems have failed, a solution is to resort to a system based on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes. This approach has been demonstrated to be successful for structural and mechanistic analyses not only for homologous production of W. succinogenes membrane proteins but also for the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni. The procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented in detail for the examples of the quinol:fumarate reductase and the SdhABE complexes of W. succinogenes. Subsequently, further projects using W. succinogenes as expression host are covered.

  11. Production of recombinant multiheme cytochromes c in Wolinella succinogenes.

    PubMed

    Kern, Melanie; Simon, Jörg

    2011-01-01

    Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c.

  12. Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

    PubMed Central

    Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.

    2015-01-01

    Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases. PMID:26629814

  13. Evaluating models of cellulose degradation by Fibrobacter succinogenes S85

    DOE PAGES

    Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.

    2015-12-02

    Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins,more » and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.« less

  14. Production of recombinant multiheme cytochromes c in Wolinella succinogenes.

    PubMed

    Kern, Melanie; Simon, Jörg

    2011-01-01

    Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c. PMID:21185447

  15. Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85.

    PubMed

    Burnet, Meagan C; Dohnalkova, Alice C; Neumann, Anthony P; Lipton, Mary S; Smith, Richard D; Suen, Garret; Callister, Stephen J

    2015-01-01

    Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases. PMID:26629814

  16. Hemolysin patterns of Actinobacillus pleuropneumoniae.

    PubMed Central

    Frey, J; Nicolet, J

    1990-01-01

    The secreted hemolytic activities produced by the reference strains and field isolates of the 12 serotypes and 2 subtypes of Actinobacillus pleuropneumoniae were analyzed. Serotype 1 produced a Ca2(+)-inducible hemolysin, which was previously characterized as a 105-kilodalton protein and was named hemolysin I (HlyI). Serotypes 2, 4, 6, 7, and 8 produced a different hemolytic activity that was not inducible by Ca2+ but required this ion for its activity. The hemolytic activity produced by these serotypes was much weaker than that found in serotype 1 and was not neutralized by rabbit antibodies against HlyI. It was, however, neutralized by serum from pigs that were experimentally infected with a serotype 2 strain and was called hemolysin II (HlyII). Serotypes 5a, 5b, 9, 10, and 11 produced both HlyI and HlyII. In these strains, HlyI was the major contributor to the hemolytic activity. The remaining serotypes, 3 and 12, produced a very weak hemolytic activity, which was not further analyzed. Immunoblot analysis of the culture supernatants from all 12 serotypes with rabbit polyclonal antibodies directed against HlyI revealed reactions with a protein in the 105-kilodalton size range for all serotypes, indicating that HlyI and HlyII might be serologically related. Strains producing active HlyI seem to belong to serotypes that are generally considered to be virulent types and that are frequently isolated from pigs in severe pleuropneumonia outbreaks. Images PMID:2312671

  17. News & notes: paper digestion by the cellulolytic ruminal bacterium Fibrobacter succinogenes.

    PubMed

    Martin, S A; Martin, J A

    1998-12-01

    The objective of this study was to evaluate the ability of the predominant cellulolytic ruminal bacterium Fibrobacter succinogenes S85 to digest filter paper, office paper, newspaper, and magazine paper. When F. succinogenes S85 was incubated with all four paper sources, little digestion of any paper occurred between 0 and 48 h. However, digestion of filter paper increased from 12% at 48 h to 80% at 120 h. No significant digestion of office paper, newspaper, or magazine paper occurred between 48 and 120 h. These results suggest that F. succinogenes S85 is unable to digest office paper, newspaper, or magazine paper.

  18. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-01-01

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  19. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-30

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  20. Identification of an anaerobic bacterium which reduces perchlorate and chlorate as Wolinella succinogenes

    SciTech Connect

    Wallace, W.; Attaway, H. |

    1995-12-31

    Perchlorate and chlorate salts are widely used by the chemical, aerospace and defense industries as oxidizers in propellant, explosives and pyrotechnics. The authors have isolated a anaerobic bacterium which is capable of the dissimilatory reduction of both perchlorate and chlorate for energy and growth. Strain HAP-1 is a gram negative, thin rod, non-sporeforming, highly motile strict anaerobe. Antibiotic resistance profiles, utilization of carbon substrates and electron acceptors demonstrated similar physiological characteristics to Wolinella succinogenes. Pairwise comparisons of 16S RNA sequences showed only a 0.75% divergence between strain HAP-1 and W. succinogenes. Physiological, morphological and 16S RRNA sequence data indicate strain HAP-1 is a subspecies of W. succinogenes that can utilize perchlorate and chlorate as terminal electron acceptors.

  1. The Complete Genome Sequence of Fibrobacter succinogenes S85 Reveals a Cellulolytic and Metabolic Specialist

    PubMed Central

    Suen, Garret; Weimer, Paul J.; Stevenson, David M.; Aylward, Frank O.; Boyum, Julie; Deneke, Jan; Drinkwater, Colleen; Ivanova, Natalia N.; Mikhailova, Natalia; Chertkov, Olga; Goodwin, Lynne A.; Currie, Cameron R.; Mead, David; Brumm, Phillip J.

    2011-01-01

    Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation. PMID:21526192

  2. Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase.

    PubMed

    Lancaster, C Roy D

    2003-11-27

    The three-dimensional structure of Wolinella succinogenes quinol:fumarate reductase (QFR), a dihaem-containing member of the superfamily of succinate:quinone oxidoreductases (SQOR), has been determined at 2.2 A resolution by X-ray crystallography [Lancaster et al., Nature 402 (1999) 377-385]. The structure and mechanism of W. succinogenes QFR and their relevance to the SQOR superfamily have recently been reviewed [Lancaster, Adv. Protein Chem. 63 (2003) 131-149]. Here, a comparison is presented of W. succinogenes QFR to the recently determined structure of the mono-haem containing succinate:quinone reductase from Escherichia coli [Yankovskaya et al., Science 299 (2003) 700-704]. In spite of differences in polypeptide and haem composition, the overall topology of the membrane anchors and their relative orientation to the conserved hydrophilic subunits is strikingly similar. A major difference is the lack of any evidence for a 'proximal' quinone site, close to the hydrophilic subunits, in W. succinogenes QFR.

  3. Cellulase and xylanase release from Bacteroides succinogenes and its importance in the rumen environment

    SciTech Connect

    Forsberg, C.W.; Beveridge, T.J.; Hellstrom, A.

    1981-11-01

    During growth of Bacteroides succinogenes in a liquid medium with cellulose as the source of carbohydrate, greater than 80% of the carboxymethylcellulase (endo-beta-1,4-glucanase), xylanase, and aryl-beta-xylosidase and 50% of the aryl-beta-glucosidase released from cells into the culture fluid. Less than 25% of the cellobiase activity was detected in the culture fluid. Approximately 50% of each of the released enzymes measured was associated with sedimentable subcellular membrane vesicles. The vesicles appeared to be released from the outer membrane of intact cells by bleb formation, primarily in pockets between the cells and the cellulose, although a few unattached cells with blebs were seen. Many vesicles were seen adhering to cellulose, and they were also seen free in the culture fluid. These data suggest that Bacteroides succinogenes releases hydrolytic enzymes in non-sedimentable and particulate forms during growth by a mechanism which has until now received little attention. Cellulose incubated in a porous nylon bag in the rumen was colonized by bacteria resembling Bacteroides succinogenes, and subcellular vesicles were seen penetrating channels and fractures in the cellulose. On this basis, it is suggested that bacteroides succinogenes cells in the rumen contribute to an extracellular population of subcellular vesicles that possess cellulolytic and hemicellulolytic activities which probably enhance polymer digestion and provide a source of sugars for microbes lacking polymer-degrading activity, thereby contributing to a stable heterogeneous microbial population. (Refs. 46).

  4. Inhibition of fibroblast proliferation by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Shenker, B J; Kushner, M E; Tsai, C C

    1982-01-01

    We have examined soluble sonic extracts of Actinobacillus actinomycetemcomitans for their ability to alter human and murine fibroblast proliferation. We found that extracts of all A. actinomycetemcomitans strains examined (both leukotoxic and nonleukotoxic) caused a dose-dependent inhibition of both murine and human fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation). Addition of sonic extract simultaneously with [3H]thymidine had no effect on incorporation, indicating that suppression was not due to the presence of excessive amounts of cold thymidine. Inhibition of DNA synthesis was also paralleled by decreased RNA synthesis ([3H]uridine incorporation) and by a decrease in cell growth as assessed by direct cell counts; there was no effect on cell viability. The suppressive factor(s) is heat labile; preliminary purification and characterization studies indicate that it is a distinct and separate moiety from other A. actinomycetemcomitans mediators previously reported, including leukotoxin, immune suppressive factor, and endotoxin. Although it is not clear how A. actinomycetemcomitans acts to cause disease, we propose that one aspect of the pathogenicity of this organism rests in its ability to inhibit fibroblast growth, which in turn could contribute to the collagen loss associated with certain forms of periodontal disease, in particular juvenile periodontitis. PMID:7152684

  5. Killing of Actinobacillus actinomycetemcomitans by human lactoferrin.

    PubMed Central

    Kalmar, J R; Arnold, R R

    1988-01-01

    Actinobacillus actinomycetemcomitans is a fastidious, facultative gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human neutrophils have demonstrated bactericidal activity against A. actinomycetemcomitans, and much of the oxygen-dependent killing has been attributed to the myeloperoxidase-H2O2-halide system. However, the contribution of other neutrophil components to killing activity is obscure. Lactoferrin, an iron-binding glycoprotein, is a major constituent of neutrophil-specific granules and is also found in mucosal secretions. In this report, we show that human lactoferrin is bactericidal for A. actinomycetemcomitans. Killing activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log decrease in viability within 120 min at 37 degrees C at a concentration of 1.9 microM. Besides exhibiting concentration dependence, killing kinetics were affected by minor variations in temperature and pH. Magnesium, a divalent cation thought to stabilize lipopolysaccharide interactions on the surface of gram-negative organisms, enhanced lactoferrin killing of A. actinomycetemcomitans, while other cations, such as potassium and calcium, had no effect. Our data suggest that lactoferrin contributes to killing of A. actinomycetemcomitans by human neutrophils and that it may also play a significant role in innate secretory defense against this potential periodontopathogen. PMID:3417349

  6. Isolation of Actinobacillus suis from a cat's lung.

    PubMed Central

    Daignault, D; Chouinard, L; Møller, K; Ahrens, P; Messier, S; Higgins, R

    1999-01-01

    Actinobacillus suis has been isolated from the lungs of 9-month-old cat. The bacterium was characterized biochemically as well as genetically, and its sensitivity profile to different antimicrobial agents was established. The role of this isolate in the cat's condition is discussed. PMID:9919368

  7. Evaluating models of cellulose degradation by Fibrobacter succinogenes S85

    SciTech Connect

    Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.

    2015-12-02

    Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.

  8. Classification of avian haemolytic Actinobacillus-like organisms (Bisgaard taxon 26) associated with anseriforme birds as Actinobacillus anseriformium sp. nov.

    PubMed

    Bisgaard, M; Christensen, H

    2012-02-01

    Avian haemolytic Actinobacillus-like organisms have tentatively been named Bisgaard taxon 26. Phenotypic information has been published on 65 strains of this taxon. In the current study, 31 isolates were selected for genotypic characterization. Thirty strains had the same rpoB sequence and only one strain diverged in 1 nt. The highest rpoB similarity to members of other taxa was 89.7 % to the type strain of Actinobacillus equuli subsp. haemolyticus and the similarity to the type strain of the type species, Actinobacillus lignieresii, was 88.2 %. The lowest 16S rRNA gene sequence similarity between strains of the group was determined in previous investigations to be 99.6 % and the highest similarities of 96.4 and 96.2 % outside the group were obtained to the reference strain of Actinobacillus genomospecies 2 and to the type strain of A. equuli subsp. equuli, respectively; 95.8-95.3 % similarity was obtained with the type strain of A. lignieresii. recN gene sequence similarities within the group were from 99.5 % (strains F66(T) and F64) to 99.8 % (strains F66(T) and F67) corresponding to genome similarities of 93.9-94.6 %, which are near the upper limit for species compared with other members of the Pasteurellaceae. The highest recN similarity outside the group (83.4 %) was observed to the type strain of Actinobacillus capsulatus, whereas the similarity to the type strain of A. lignieresii was 80.9 %, corresponding to genome similarities of 57.7 and 52.0 %, respectively. All isolates meet the phenotypic characters outlined for Actinobacillus (urease-, phosphatase- and porphyrin-positive, indole-negative, acid production from fructose, sucrose, maltose and dextrin). β-Haemolysis of bovine blood is observed and isolates may demonstrate in vitro satellitic growth, referred to as V-factor or NAD requirement. Isolates have been obtained from the upper respiratory tract of web-footed birds in which they may cause sinusitis, conjunctivitis and

  9. Production and consumption of nitrous oxide in nitrate-ammonifying Wolinella succinogenes cells.

    PubMed

    Luckmann, Monique; Mania, Daniel; Kern, Melanie; Bakken, Lars R; Frostegård, Asa; Simon, Jörg

    2014-08-01

    Global warming is moving more and more into the public consciousness. Besides the commonly mentioned carbon dioxide and methane, nitrous oxide (N2O) is a powerful greenhouse gas in addition to its contribution to depletion of stratospheric ozone. The increasing concern about N2O emission has focused interest on underlying microbial energy-converting processes and organisms harbouring N2O reductase (NosZ), such as denitrifiers and ammonifiers of nitrate and nitrite. Here, the epsilonproteobacterial model organism Wolinella succinogenes is investigated with regard to its capacity to produce and consume N2O during growth by anaerobic nitrate ammonification. This organism synthesizes an unconventional cytochrome c nitrous oxide reductase (cNosZ), which is encoded by the first gene of an atypical nos gene cluster. However, W. succinogenes lacks a nitric oxide (NO)-producing nitrite reductase of the NirS- or NirK-type as well as an NO reductase of the Nor-type. Using a robotized incubation system, the wild-type strain and suitable mutants of W. succinogenes that either produced or lacked cNosZ were analysed as to their production of NO, N2O and N2 in both nitrate-sufficient and nitrate-limited growth medium using formate as electron donor. It was found that cells growing in nitrate-sufficient medium produced small amounts of N2O, which derived from nitrite and, most likely, from the presence of NO. Furthermore, cells employing cNosZ were able to reduce N2O to N2. This reaction, which was fully inhibited by acetylene, was also observed after adding N2O to the culture headspace. The results indicate that W. succinogenes cells are competent in N2O and N2 production despite being correctly grouped as respiratory nitrate ammonifiers. N2O production is assumed to result from NO detoxification and nitrosative stress defence, while N2O serves as a terminal electron acceptor in anaerobic respiration. The ecological implications of these findings are discussed. PMID:24781903

  10. Production and consumption of nitrous oxide in nitrate-ammonifying Wolinella succinogenes cells.

    PubMed

    Luckmann, Monique; Mania, Daniel; Kern, Melanie; Bakken, Lars R; Frostegård, Asa; Simon, Jörg

    2014-08-01

    Global warming is moving more and more into the public consciousness. Besides the commonly mentioned carbon dioxide and methane, nitrous oxide (N2O) is a powerful greenhouse gas in addition to its contribution to depletion of stratospheric ozone. The increasing concern about N2O emission has focused interest on underlying microbial energy-converting processes and organisms harbouring N2O reductase (NosZ), such as denitrifiers and ammonifiers of nitrate and nitrite. Here, the epsilonproteobacterial model organism Wolinella succinogenes is investigated with regard to its capacity to produce and consume N2O during growth by anaerobic nitrate ammonification. This organism synthesizes an unconventional cytochrome c nitrous oxide reductase (cNosZ), which is encoded by the first gene of an atypical nos gene cluster. However, W. succinogenes lacks a nitric oxide (NO)-producing nitrite reductase of the NirS- or NirK-type as well as an NO reductase of the Nor-type. Using a robotized incubation system, the wild-type strain and suitable mutants of W. succinogenes that either produced or lacked cNosZ were analysed as to their production of NO, N2O and N2 in both nitrate-sufficient and nitrate-limited growth medium using formate as electron donor. It was found that cells growing in nitrate-sufficient medium produced small amounts of N2O, which derived from nitrite and, most likely, from the presence of NO. Furthermore, cells employing cNosZ were able to reduce N2O to N2. This reaction, which was fully inhibited by acetylene, was also observed after adding N2O to the culture headspace. The results indicate that W. succinogenes cells are competent in N2O and N2 production despite being correctly grouped as respiratory nitrate ammonifiers. N2O production is assumed to result from NO detoxification and nitrosative stress defence, while N2O serves as a terminal electron acceptor in anaerobic respiration. The ecological implications of these findings are discussed.

  11. First isolation of Actinobacillus genomospecies 2 in Japan

    PubMed Central

    MURAKAMI, Miyuki; SHIMONISHI, Yoshimasa; HOBO, Seiji; NIWA, Hidekazu; ITO, Hiroya

    2015-01-01

    We describe here the first isolation of Actinobacillus genomospecies 2 in Japan. The isolate was found in a septicemic foal and characterized by phenotypic and genetic analyses, with the latter consisting of 16S rDNA nucleotide sequence analysis plus multilocus sequence analysis using three housekeeping genes, recN, rpoA and thdF, that have been proposed for use as a genomic tool in place of DNA-DNA hybridization. PMID:26668165

  12. Bacteriocin production by Actinobacillus actinomycetemcomitans isolated from the oral cavity of humans with periodontal disease, periodontally healthy subjects and marmosets.

    PubMed

    Lúcia, Lima Francisca; Farias, Flávio F; Eustáquio, Costa José; Auxiliadora, Maria; Carvalho, R; Alviano, Celuta S; Farias, Luiz M

    2002-01-01

    The ability of Actinobacillus actinomycetemcomitans to produce bacteriocin has rarely been reported. Antagonistic substance production may confer an important ecological advantage for the producer microorganisms, especially in a competitive ecosystem such as the oral cavity. In the present study, 75 A. actinomycetemcomitans strains isolated from the oral cavity of human patients with periodontal disease, periodontally healthy subjects and marmosets, as well as two reference strains (A. actinomycetemcomitans ATCC 29523 and FDC Y4) were evaluated for auto-, iso-, and heteroantagonistic activity. Fifty-one (68.00%) strains exhibited antagonistic activity; heteroantagonism was observed more often than isoantagonism. Isolated strains antagonized 17 different species of gram-positive and gram-negative bacteria from the oral and nonoral microbiota. Sensitivity to heat and to proteolytic enzymes constituted strong evidence that the antagonistic substance has a proteic nature. Taken together, our data enabled us to confirm that the antagonistic substance detected was a bacteriocin. The wide spectrum of activity indicates the possibility that more than one antagonistic substance is produced and that these substances play an important role in the ecological balance of the oral ecosystem.

  13. Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide.

    PubMed

    Wilson, M E; Schifferle, R E

    1991-04-01

    A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b-specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism.

  14. Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

    PubMed Central

    Matte, A; Forsberg, C W

    1992-01-01

    Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo. Images PMID:1539970

  15. Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

    PubMed Central

    Raut, Mahendra P.; Karunakaran, Esther; Mukherjee, Joy; Biggs, Catherine A.; Wright, Phillip C.

    2015-01-01

    Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for

  16. Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein.

    PubMed

    Simon, Jörg; Sänger, Monica; Schuster, Stephan C; Gross, Roland

    2003-07-01

    The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from

  17. Immune response to an Actinobacillus pleuropneumoniae vaccine in swine

    PubMed Central

    Rousseau, Paul; Assaf, Robert; Boulay, Gaston; Désy, Marie

    1988-01-01

    Piglets vaccinated with an inactivated tetravalent vaccine (Pleurovac 4) against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7, produced circulating antibodies after a first intramuscular injection and showed an anamnestic reaction after a second. The rise in antibody levels in vaccinated piglets was statistically significant when compared with those of the control group. The administration of 1 or 2 mL of vaccine did not lead to significantly different antibody levels. The specificity of the immune response is demonstrated by an increase in titer to all four serotypes in pigs given the tetravalent vaccine, but an increase in titer to only two serotypes in pigs given a bivalent vaccine (Pleurovac). PMID:17423199

  18. A longitudinal microbiological investigation of Actinobacillus actinomycetemcomitans and Eikenella corrodens in juvenile periodontitis.

    PubMed Central

    Mandell, R L

    1984-01-01

    Longitudinal clinical and microbiological monitoring of subjects with localized juvenile periodontitis indicated that Actinobacillus actinomycetemcomitans and Eikenella corrodens were significantly associated (P less than 0.05) with active tissue destruction. PMID:6381313

  19. Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria. [Ruminococcus flavefaciens, R. albus, Bacteroides succinogenes

    SciTech Connect

    Hiltner, P.; Dehority, B.A.

    1983-09-01

    The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria. 30 references.

  20. Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed

    Braunthal, S D; Holt, S C; Tanner, A C; Socransky, S S

    1980-06-01

    Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content. PMID:7430333

  1. Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed Central

    Braunthal, S D; Holt, S C; Tanner, A C; Socransky, S S

    1980-01-01

    Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content. PMID:7430333

  2. Actinobacillus actinomycetemcomitans contamination of toothbrushes from patients harbouring the organism.

    PubMed

    Müller, H P; Lange, D E; Müller, R F

    1989-07-01

    The main ecological niche of Actinobacillus actinomycetemcomitans (A.a.) seems to be the periodontal pocket, but it can also be isolated from supragingival plaque, buccal and tongue mucosa, or saliva. We examined toothbrushes from 21 patients, all identified as harbouring moderate to large numbers of A.a. in subgingival plaque, for contamination with this organism. 29% of the toothbrushes presented by our patients yielded detectable numbers of A.a. Immediately after toothbrushing this figure rose to 62%, but dropped to 50% after 1 h. Numbers of isolated A.a. on toothbrushes were weakly correlated with the degree of periodontal destruction, and significantly more numbers of A.a. on toothbrushes could be detected if the organism was found on mucous membranes or in saliva. There was no association with gingival inflammation, supragingival plaque nor mean numbers of isolated subgingival A.a. PMID:2760252

  3. The antibacterial mechanism of berberine against Actinobacillus pleuropneumoniae.

    PubMed

    Kang, Shuai; Li, Zhengwen; Yin, Zhongqiong; Jia, Renyong; Song, Xu; Li, Li; Chen, Zhenzhen; Peng, Lianci; Qu, Jing; Hu, Zhiqiang; Lai, Xin; Wang, Guangxi; Liang, Xiaoxia; He, Changliang; Yin, Lizi

    2015-01-01

    This study demonstrated berberine to be a potential natural compound against Actinobacillus pleuropneumoniae. Liquid doubling dilution, transmission electron microscopy (TEM), SDS-PAGE and 4',6-diamidino-2-phenylindole (DAPI) staining were employed to elucidate the antibacterial activity and mechanism of berberine. The minimal inhibitory concentration of berberine was 0.3125 mg/mL, and time-kill curves showed concentration and time dependence. The TEM micrographs displayed damaged cell wall, concentrated cytoplasm, cytoplasmic content leakage and cell death. SDS-PAGE and DAPI assays revealed that berberine can restrain DNA and protein syntheses. Berberine inhibited the synthesis of proteins associated with the growth and cleavage of bacteria and then blocked the division and development of bacteria. The compound ultimately induced cytoplasm pyknosis and bacterial death.

  4. Minimal inhibitory concentrations of antimicrobial agents against Actinobacillus pleuropneumoniae.

    PubMed Central

    Nadeau, M; Larivière, S; Higgins, R; Martineau, G P

    1988-01-01

    Forty-five isolates of Actinobacillus pleuropneumoniae were tested for susceptibility to 12 antimicrobial agents using a microdilution method for the minimal inhibitory concentration determinations. These results confirmed the high prevalence of A. pleuropneumoniae strains resistant to antibiotics as reported earlier using the disc diffusion method (Kirby-Bauer method). While 36% of the isolates were resistant to the penicillins, 47% were resistant to chloramphenicol and 68% were resistant to tetracycline. Minimal inhibitory concentrations for the resistant isolates were approximately 32 times higher than those for the susceptible isolates to the above antibacterial agents. The isolates were in general weakly susceptible or resistant to spectinomycin, lincomycin, tiamulin and spiramycin whereas most of them were susceptible to gentamicin, trimethoprim and erythromycin. The susceptibility pattern was similar throughout the 1980 to 1984 period. The 14 serotype 5 isolates were more resistant to tetracycline but less resistant to chloramphenicol and the penicillins than the 28 serotype 1 isolates. PMID:3167716

  5. Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs.

    PubMed Central

    Jobert, J L; Savoye, C; Cariolet, R; Kobisch, M; Madec, F

    2000-01-01

    In order to demonstrate the possible role of aerosol in the transmission of Actinobacillus pleuropneumoniae, an experiment including 18 specific pathogen-free (SPF), 10-week-old piglets, randomly distributed into 2 adjacent units, was carried out. In these facilities, air was forced through absolute filters to prevent any contact with infectious agents. During the first 6 d post inoculation, the 2 units were connected by a rectangular opening and the air circulation was forced by the ventilation system from unit A (inoculated pigs) to unit B (non-inoculated pigs). The A. pleuropneumoniae strain (biovar 1 serovar 9) was isolated in France from an outbreak of porcine pleuropneumonia. Two different infecting doses, 10(7) cfu/animal and 10(8) cfu/animal, were inoculated by intranasal route in 6 pigs of unit A. The infection spread quickly from the inoculated pigs to the non-inoculated pigs. Clinical signs were acute during the 4 d post inoculation: hyperthermia, respiratory distress and, sometimes, death (6 pigs of the unit A and 2 pigs of the unit B). All pigs seroconverted against A. pleuropneumoniae serovar 9 within 2 weeks. Lung lesions were severe: fibrinous pleurisy and lung hemorrhages in the acute stage, pleural adherences and focal pulmonary necrosis in the chronic stage. Actinobacillus pleuropneumoniae was isolated from the tonsils and/or lungs in 16 animals. It could be also isolated from the air of the experimental unit. This study showed that A. pleuropneumoniae was readily transmitted through aerosol over a distance of at least 2.5 m. Images Figure 1. Figure 2. PMID:10680652

  6. Biosynthetic Pathways of Vibrio succinogenes growing with fumarate as terminal electron acceptor and sole carbon source.

    PubMed

    Bronder, M; Mell, H; Stupperich, E; Kröger, A

    1982-05-01

    1. With fumarate as the terminal electron acceptor and either H2 or formate as donor, Vibrio succinogenes could grow anaerobically in a mineral medium using fumarate as the sole carbon source. Both the growth rate and the cell yield were increased when glutamate was also present in the medium. 2. Glutamate was incorporated only into the amino acids of the glutamate family (glutamate, glutamine, proline and arginine) of the protein. The residual cell constituents were synthesized from fumarate. 3. Pyruvate and phosphoenolpyruvate, as the central intermediates of most of the cell constituents, were formed through the action of malic enzyme and phosphoenolpyruvate synthetase. Fructose-1,6-bisphosphate aldolase was present in the bacterium suggesting that this enzyme is involved in carbohydrate synthesis. 4. In the absence of added glutamate the amino acids of the glutamate family were synthesized from fumarate via citrate. The enzymes involved in glutamate synthesis were present. 5. During growth in the presence of glutamate, net reducing equivalents were needed for cell synthesis. Glutamate and not H2 or formate was used as the source of these reducing equivalents. For this purpose part of the glutamate was oxidized to yield succinate and CO2. 6. The alpha-ketoglutarate dehydrogenase involved in this reaction was found to use ferredoxin as the electron acceptor. The ferredoxin of the bacterium was reoxidized by means of a NADP-ferredoxin oxidoreductase. Enzymes catalyzing the reduction of NAD, NADP or ferredoxin by H2 or formate were not detected in the bacterium. PMID:7103660

  7. Application of a data reconciliation method to the stoichiometric analysis of Fibrobacter succinogenes growth.

    PubMed

    Guiavarch, Erell; Pons, Agnes; Creuly, Catherine; Dussap, Claude-Gilles

    2008-12-01

    Fibrobacter succinogenes S85, a strictly anaerobic Gram-negative bacterium, was grown in continuous culture in a bioreactor at different dilution rates (0.02 to 0.092 h(-1)) on a fully synthetic culture medium with glucose as carbon source. Glucose and ammonium sulfate consumption, as well as biomass, succinate, acetate, formate, and carbohydrate production were regularly measured. The relevant biomass elemental compositions were established for each dilution rate. Robustness of the experimental information was checked by C and N mass balances estimation, which were satisfactory. A detailed overall stoichiometry analysis of the process, including all substrates and products of the culture, was proposed. Online and off-line parameters measured during the culture brought a large number of data which were weighted by their respective variance associated to the measured value. The material balance resulted in an overdetermined linear system of equations made of weighted relationships including experimental data, elemental balances (C, H, O, N, S, Na), and an additional constraint. The mass balances involved in stoichiometric equations were solved using data reconciliation and linear algebra methods to take into account error measurements. This methodology allowed to establish the overall stoichiometric equation for each dilution rate studied.

  8. Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms.

    PubMed Central

    MacInnes, J I; Rosendal, S

    1987-01-01

    Outer membrane protein (OMP)-enriched extracts and whole-cell protein preparations of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms were examined by polyacrylamide gel electrophoresis and immunoblotting. Both the OMP-enriched and whole-cell protein profiles of Actinobacillus suis, A. pleuropneumoniae (NAD-independent biovar), A. lignieresii, and Pasteurella haemolytica were very similar to those of H. pleuropneumoniae serotypes 1 to 8. Antisera prepared against H. pleuropneumoniae typically recognized three major OMP antigens with approximate molecular weights of 17,000 (17K), 32K, and 42K in immunoblots of H. pleuropneumoniae serotypes 1 to 8, Actinobacillus spp., and P. haemolytica. Antisera prepared against Actinobacillus spp. and Haemophilus sp. "minor group" also recognized these 17K, 32K, and 42K antigens. Using absorbed sera, we demonstrated that the 17K antigen had an epitope (or epitopes) common to all the gram-negative organisms examined, including Escherichia coli. The 32K and 42K antigens had epitopes common to members of the family Pasteurellaceae but, in the case of the 32K antigen, also contained unique epitopes. These results provide a basis for understanding the lack of specificity of serodiagnostic tests for H. pleuropneumoniae infection and provide another line of evidence for the association of H. pleuropneumoniae with the genus Actinobacillus. Images PMID:3298061

  9. Biochemical studies of Klebsiella pneumoniae NifL reduction using reconstituted partial anaerobic respiratory chains of Wolinella succinogenes.

    PubMed

    Thummer, Robert; Klimmek, Oliver; Schmitz, Ruth A

    2007-04-27

    In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK(6)) or 8-methylmenaquinone (MMK(6)) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK(6) from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.

  10. Genome Sequence of Propionibacterium acidipropionici ATCC 55737.

    PubMed

    Luna-Flores, Carlos H; Nielsen, Lars K; Marcellin, Esteban

    2016-01-01

    Propionibacterium acidipropionici produces propionic acid as its main fermentation product. Traditionally derived from fossil fuels, environmental and sustainable issues have revived the interest in producing propionic acid using biological resources. Here, we present the closed sequence of Propionibacterium acidipropionici ATCC 55737, an efficient propionic acid producer. PMID:27198010

  11. Cell Yields of Vibrio succinogenes growing with formate and fumarate as sole carbon and energy sources in chemostat culture.

    PubMed

    Mell, H; Bronder, M; Kröger, A

    1982-05-01

    Vibrio succinogenes which gains all the ATP by anaerobic electron transport phosphorylation, was grown in continuous culture on a defined medium with formate and fumarate as sole energy sources. The growth yield at infinite dilution rate (Ymax) was obtained by extrapolation from the growth yields measured at various dilution rates. With formate as the growth limiting substrate, Ymax was found as 14 g dry cells/mol formate. Under these conditions growth was limited by the rate of energy supply, because formate is used only as a catabolic substrate (Bronder et al. 1982). The YmaxATP calculated from the ATP requirement for cell synthesis was 18 g dry cells/mol ATP. This gives an ATP/2e ratio of 0.8. The ATP/2e ratio in vitro had been measured as 1 (Kröger and Winkler 1981). It is concluded that growing V. succinogenes gain at least 80% the stoichiometrically possible amount of ATP, when growth is limited by energy supply. PMID:7103661

  12. Immunosuppressive factor from Actinobacillus actinomycetemcomitans down regulates cytokine production.

    PubMed Central

    Kurita-Ochiai, T; Ochiai, K

    1996-01-01

    A cytoplasmic soluble fraction of Actinobacillus actinomycetemcomitans Y4 was isolated and characterized as suppressing mitogen-stimulated proliferation of and cytokine production by C3H/HeN mouse splenic T cells. This factor, designated suppressive factor 1 (SF1), was isolated from the supernatant of sonicated whole bacteria and purified by Q-Sepharose Fast Flow column chromatography, DEAE-Sepharose Fast Flow column chromatography, hydroxyapatite high-pressure liquid chromatography (HPLC), and Protein Pack 300 & 125 gel filtration HPLC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified SF1 migrated as a single band corresponding to a molecular mass of 14 kDa. This molecule was protease labile, heat resistant, and noncytotoxic. N'-terminal sequence analysis revealed no homology with any known peptides of periodontopathic bacteria or with any host-derived growth factors. Purified SF1 suppressed the proliferation of mouse splenic T cells which had been stimulated with concanavalin A, as well as suppressing the production of interleukin-2 (IL-2), gamma interferon, IL-4, and IL-5 from CD4+ T cells as 0.1 microgram/ml or more. These data suggest that SF1 produced by the periodontal pathogen A. actinomycetemcomitans functions as a virulence factor by down regulating T-cell proliferation and cytokine production at local defense sites. PMID:8557373

  13. Antibiofilm activity of Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide.

    PubMed

    Karwacki, Michael T; Kadouri, Daniel E; Bendaoud, Meriem; Izano, Era A; Sampathkumar, Vandana; Inzana, Thomas J; Kaplan, Jeffrey B

    2013-01-01

    Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications. PMID:23691104

  14. Antibiofilm Activity of Actinobacillus pleuropneumoniae Serotype 5 Capsular Polysaccharide

    PubMed Central

    Karwacki, Michael T.; Kadouri, Daniel E.; Bendaoud, Meriem; Izano, Era A.; Sampathkumar, Vandana; Inzana, Thomas J.; Kaplan, Jeffrey B.

    2013-01-01

    Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications. PMID:23691104

  15. Identification of Fur-regulated genes in Actinobacillus actinomycetemcomitans.

    PubMed

    Haraszthy, Violet I; Jordan, Shawn F; Zambon, Joseph J

    2006-03-01

    Actinobacillus actinomycetemcomitans is an oral pathogen that causes aggressive periodontitis as well as sometimes life-threatening, extra-oral infections. Iron regulation is thought to be important in the pathogenesis of A. actinomycetemcomitans infections and, consistent with this hypothesis, the fur gene has recently been identified and characterized in A. actinomycetemcomitans. In this study, 14 putatively Fur-regulated genes were identified by Fur titration assay (Furta) in A. actinomycetemcomitans, including afuA, dgt, eno, hemA, tbpA, recO and yfe - some of which are known to be Fur regulated in other species. A fur mutant A. actinomycetemcomitans strain was created by selecting for manganese resistance in order to study the Fur regulon. Comparisons between the fur gene sequences revealed that nucleotide 66 changed from C in the wild-type to T in the mutant strain, changing leucine to isoleucine. The fur mutant strain expressed a nonfunctional Fur protein as determined by Escherichia coli-based ferric uptake assays and Western blotting. It was also more sensitive to acid stress and expressed higher levels of minC than the wild-type strain. minC, which inhibits cell division in other bacterial species and whose regulation by iron has not been previously described, was found to be Fur regulated in A. actinomycetemcomitans by Furta, by gel shift assays, and by RT-qPCR assays for gene expression. PMID:16514158

  16. Detection of Actinobacillus pleuropneumoniae in drinking water from pig farms.

    PubMed

    Loera-Muro, Victor M; Jacques, Mario; Tremblay, Yannick D N; Avelar-González, Francisco J; Loera Muro, Abraham; Ramírez-López, Elsa M; Medina-Figueroa, Alejandra; González-Reynaga, Higinio M; Guerrero-Barrera, Alma L

    2013-03-01

    Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia and is normally transmitted by aerosols and direct contact between animals. A. pleuropneumoniae has traditionally been considered an obligate pathogen of pigs and its presence in the environment has yet to be investigated. Here, the presence of A. pleuropneumoniae was detected in drinking water of pig farms in Mexico using a PCR specific for the RTX toxin gene, apxIV. The presence of A. pleuropneumoniae in farm drinking water was confirmed by indirect immunofluorescence using an A. pleuropneumoniae-specific polyclonal antibody and by fluorescent in situ hybridization. Viable bacteria from the farm drinking water were detected using the Live/Dead BacLight stain. Additionally, viable A. pleuropneumoniae was selected and isolated using the cAMP test and the identity of the isolated bacteria were confirmed by Gram staining, a specific polyclonal antibody and an A. pleuropneumoniae-specific PCR. Furthermore, biofilms were observed by scanning electron microscopy in A. pleuropneumoniae-positive samples. In conclusion, our data suggest that viable A. pleuropneumoniae is present in the drinking water of swine farms and may use biofilm as a strategy to survive in the environment.

  17. Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo.

    PubMed Central

    Huang, H; Potter, A A; Campos, M; Leighton, F A; Willson, P J; Yates, W D

    1998-01-01

    To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions. PMID:9553707

  18. Lytic sensitivity of Actinobacillus actinomycetemcomitans Y4 to lysozyme.

    PubMed Central

    Iacono, V J; Boldt, P R; MacKay, B J; Cho, M I; Pollock, J J

    1983-01-01

    The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be

  19. Effects of growth conditions on biofilm formation by Actinobacillus pleuropneumoniae

    PubMed Central

    Labrie, Josée; Pelletier-Jacques, Geneviève; Deslandes, Vincent; Ramjeet, Mahendrasingh; Auger, Eliane; Nash, John H.E.; Jacques, Mario

    2010-01-01

    Biofilm formation is an important virulence trait of many bacterial pathogens. It has been reported in the literature that only two of the reference strains of the swine pathogen Actinobacillus pleuropneumoniae, representing serotypes 5b and 11, were able to form biofilm in vitro. In this study, we compared biofilm formation by the serotype 1 reference strain S4074 of A. pleuropneumoniae grown in five different culture media. We observed that strain S4074 of A. pleuropneumoniae is able to form biofilms after growth in one of the culture conditions tested brain heart infusion (BHI medium, supplier B). Confocal laser scanning microscopy using a fluorescent probe specific to the poly-N-acetylglucosamine (PGA) polysaccharide further confirmed biofilm formation. In accordance, biofilm formation was susceptible to dispersin B, a PGA hydrolase. Transcriptional profiles of A. pleuropneumoniae S4074 following growth in BHI-B, which allowed a robust biofilm formation, and in BHI-A, in which only a slight biofilm formation was observed, were compared. Genes such as tadC, tadD, genes with homology to autotransporter adhesins as well as genes pgaABC involved in PGA biosynthesis and genes involved in zinc transport were up-regulated after growth in BHI-B. Interestingly, biofilm formation was inhibited by zinc, which was found to be more present in BHI-A (no or slight biofilm) than in BHI-B. We also observed biofilm formation in reference strains representing serotypes 3, 4, 5a, 12 and 14 as well as in 20 of the 37 fresh field isolates tested. Our data indicate that A. pleuropneumoniae has the ability to form biofilms under appropriate growth conditions and transition from a biofilm-positive to a biofilm-negative phenotype was reversible. PMID:19737507

  20. Characterization of leukotoxin from a clinical strain of Actinobacillus actinomycetemcomitans.

    PubMed

    Diaz, Roger; Ghofaily, Lourdes Al; Patel, Jigna; Balashova, Nataliya V; Freitas, Anna C; Labib, Irene; Kachlany, Scott C

    2006-02-01

    Actinobacillus actinomycetemcomitans is a Gram negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP), a rapidly progressing and severe disease of the oral cavity that affects predominantly adolescents. A. actinomycetemcomitans is also found in extraoral infections including infective endocarditis. As one of its many virulence determinants, A. actinomycetemcomitans produces the RTX (repeats in toxin) exotoxin, leukotoxin (LtxA). LtxA specifically kills leukocytes of humans and Old World Monkeys. All of our current knowledge of A. actinomycetemcomitans LtxA is based on the protein from strain JP2, a nonadherent laboratory isolate. Because laboratory isolates can lose virulence properties, we wished to examine LtxA from a clinical isolate, NJ4500. We show that localization patterns of LtxA do not differ between the strains. Subcellular localization studies with NJ4500 revealed that LtxA localizes to the outer membrane and that the interaction between LtxA and the surface of cells is specific. Surface localized LtxA was not removed with NaCl treatment and protease protection experiments revealed that approximately 10 kDa of LtxA is exposed. We purified secreted LtxA from NJ4500 and found that the specific activity of this toxin was greater than that of secreted LtxA from JP2. For other RTX toxins, fatty acid modification affects toxin activity, and A. actinomycetemcomitans LtxA is predicted to be modified. We show by two-dimensional gel electrophoresis that NJ4500 LtxA is more highly modified than JP2 LtxA, suggesting that the difference in activities could be due to differential modification. Studies of A. actinomycetemcomitans pathogenesis should therefore consider LtxA from clinical isolates.

  1. Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans

    PubMed Central

    Tan, Kai Soo; Ong, Grace; Song, Keang Peng

    2005-01-01

    In eukaryotic cells, genes are interrupted by intervening sequences called introns. Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA. However, introns are rarely found in bacteria. Actinobacillus actinomycetemcomitans is a periodontal pathogen implicated in aggressive forms of periodontal disease. This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G2/M phase of the cell cycle. In this study, we report the presence of introns within the cdt gene of A. actinomycetemcomitans. By use of reverse transcription-PCR, cdt transcripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected. In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA. Similar results were obtained when a plasmid carrying cdt was cloned into Escherichia coli. Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3. Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1. Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur. The catalytic region of the cdt RNA was located within the cdtC gene. This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in both cis and trans configurations. PMID:15629928

  2. The gingival immune response to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

    PubMed

    Hall, E R; Falkler, W A; Martin, S A; Suzuki, J B

    1991-12-01

    The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients. PMID:1765941

  3. Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482.

    PubMed

    Wakarchuk, Warren W; Brochu, Denis; Foote, Simon; Robotham, Anna; Saxena, Hirak; Erak, Tamara; Kelly, John

    2016-01-01

    The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.

  4. Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482

    PubMed Central

    Wakarchuk, Warren W.; Brochu, Denis; Foote, Simon; Robotham, Anna; Saxena, Hirak; Erak, Tamara; Kelly, John

    2016-01-01

    The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization. PMID:26950732

  5. RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains.

    PubMed Central

    Beck, M; van den Bosch, J F; Jongenelen, I M; Loeffen, P L; Nielsen, R; Nicolet, J; Frey, J

    1994-01-01

    Actinobacillus pleuropneumoniae serotype reference strains and 204 A. pleuropneumoniae field strains representing all 12 serotypes and both biovars 1 and 2, obtained from laboratories from various countries worldwide, were analyzed for the presence of the toxin genes apxIC, apxIA, apxIB, apxID, apxIIC, apxIIA, apxIIIC, apxIIIA, apxIIIB, and apxIIID by DNA-DNA hybridization with specific gene probes. Expression of the toxins ApxI, ApxII, and ApxIII was assessed by immunoblot analysis with monoclonal antibodies. The results show that the patterns of apx genes and those of the expressed Apx toxins in biovar 1 field strains are the same as those of the genes and toxins of corresponding serotype reference strain. We found only three strains which had certain apx genes missing compared with the genes in their serotype reference strains. Analysis of the expression of the three toxins showed that nearly all strains expressed their apx genes and produced the same Apx toxins as their serotype reference strain. We found only one strain that did not produce ApxI, although it contained the apxICABD genes, and one strain which did not express ApxII but which contained apxIICA. Several field strains which initially showed that their serotype did not correspond to the apx gene profile of the reference strain and which had an unexpected virulence for the given serotype revealed that their initial serotyping was erroneous. We show that the apx gene profiles are inherent to a given serotype. The method cannot differentiate between all 12 serotypes. However, it allowed us to distinguish five groups of toxin gene patterns which showed pathological, toxicological, and epidemiological significance. None of the biovar 2 strains contained apxIII genes. The apxI and apxII genes in the biovar 2 strains, however, were the same as those found in the serotype reference strains of biovar 1. PMID:7852567

  6. Blocking enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serotype 2.

    PubMed Central

    Nielsen, R; Plambeck, T; Foged, N T

    1991-01-01

    A blocking enzyme-linked immunosorbent assay (ELISA), based upon a polyclonal rabbit antiserum specific to Actinobacillus pleuropneumoniae serotype 2, was developed for the detection of antibodies to A. pleuropneumoniae serotype 2 in pigs. By testing sera from pigs experimentally infected with the 11 recognized serotypes of A. pleuropneumoniae, the assay was proven to be specific for A. pleuropneumoniae serotype 2. With field sera from herds infected with A. pleuropneumoniae serotype 2, the assay was found to be more sensitive than the complement fixation test. Positive results were not observed with field sera from herds known to be free from Actinobacillus infection or with sera from two herds infected with either A. pleuropneumoniae serotype 6 or 8. The high diagnostic sensitivity and specificity of the blocking ELISA will make it useful in field diagnostic work. PMID:1890179

  7. Prevalence of seroreactors to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae in swine herds.

    PubMed Central

    Devenish, J; Rosendal, S; Bossé, J T; Wilkie, B N; Johnson, R

    1990-01-01

    A rabbit homologous polyclonal antiserum to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype 1 strain CM-5 was specifically produced and used in an antigen capture enzyme-linked immunosorbent assay (ELISA) to detect swine serum antibodies to this potentially important virulence factor. Sera from pigs experimentally infected with the most common disease-producing serotypes (serotypes 1, 2, 5, and 7) of A. pleuropneumoniae produced positive results in this ELISA. Of 144 serum samples collected from 10 herds free of pleuropneumonia and 155 serum samples from 11 herds with a history of the disease, 68 (47%) and 148 (95%), respectively, were found positive by the ELISA. In addition, pigs naturally infected with Actinobacillus suis produced antibodies which seroreacted in this ELISA. The results indicated that a high proportion of swine have antibodies seroreactive with the 104-kilodalton hemolysin produced by A. pleuropneumoniae. PMID:2332472

  8. Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed Central

    Holt, S C; Tanner, A C; Socransky, S S

    1980-01-01

    Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells. Images Fig. 6 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 PMID:7439996

  9. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae.

    PubMed

    Bossé, Janine T; Li, Yanwen; Atherton, Tom G; Walker, Stephanie; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Holden, Matthew T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-08-01

    The complete nucleotide sequence of a 7.7kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally. PMID:26049592

  10. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae

    PubMed Central

    Bossé, Janine T; Li, Yanwen; Atherton, Tom G; Walker, Stephanie; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Holden, Matthew TG; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-01-01

    The complete nucleotide sequence of a 7.7 kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally. PMID:26049592

  11. Draft Genome Sequence of Mycobacterium brumae ATCC 51384

    PubMed Central

    D'Auria, Giuseppe

    2016-01-01

    Here, we report the draft genome sequence of Mycobacterium brumae type strain ATCC 51384. This is the first draft genome sequence of M. brumae, a nonpathogenic, rapidly growing, nonchromogenic mycobacterium, with immunotherapeutic capacities. PMID:27125480

  12. Draft Genome Sequence of Mycobacterium brumae ATCC 51384.

    PubMed

    D'Auria, Giuseppe; Torrents, Eduard; Luquin, Marina; Comas, Iñaki; Julián, Esther

    2016-01-01

    Here, we report the draft genome sequence of Mycobacterium brumae type strain ATCC 51384. This is the first draft genome sequence of M. brumae, a nonpathogenic, rapidly growing, nonchromogenic mycobacterium, with immunotherapeutic capacities. PMID:27125480

  13. Genome sequence of the fish pathogen Flavobacterium columnare ATCC 49512

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare is a Gram-negative, rod shaped, motile, and highly prevalent fish pathogen causing columnaris disease in freshwater fish worldwide. Here, we present the complete genome sequence of F. columnare strain ATCC 49512. ...

  14. In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85.

    PubMed

    Schwaab, V; Matheron, C; Delort, A M; Gaudet, G; Forano, E

    2001-09-01

    Sodium gradients (DeltapNa) were measured in resting cells of Fibrobacter succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5- [thulium(III) 1,4,7,10-tetraazacyclododecane-N',N",N"'-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a DeltapNa of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).

  15. Influence of Serum and Glucose Additives on Survival of Actinobacillus pleuropneumoniae Aerosolized from the Freeze-Dried State.

    PubMed

    Hensel, A

    1994-06-01

    Serum and/or glucose added to Actinobacillus pleuropneumoniae suspensions before freeze-drying significantly increased survival rates of bacteria in aerosols. Aerosols with predictable numbers of viable bacteria can be made as required in an aerosol infection model. Sucrose supplementation of impinger fluids increased recovery of viable A. pleuropneumoniae.

  16. The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 14

    PubMed Central

    ITO, Hiroya

    2015-01-01

    The genetic organization of the gene involved in the capsular polysaccharide (CPS) biosynthesis of Actinobacillus pleuropneumoniae serotype 14 has been determined. The DNA region for the CPS biosynthesis of serotype 14 (cps14) comprised 9 open reading frames, designated as cps14AB1B2B3CDEFG genes, encoding Cps14A to Cps14G protein, respectively. Cps14A was similar to CpsA of A. pleuropneumoniae serotypes 1, 4 and 12; the Cps14B1 and Cps14B2 were similar to CpsB of A. pleuropneumoniae serotypes 1, 4 and 12, suggesting that CPS structure of A. pleuropneumoniae serotype 14 would belong to Group I including A. pleuropneumoniae serotypes 1, 4, 12 and 15. Surprisingly, the overall nucleotide sequence, deduced amino acid sequence, and the genetic organization of the cps14 were nearly identical to those of Actinobacillus suis. This study will provide the molecular basic knowledge for development of diagnostics and vaccine of A. pleuropneumoniae serotype 14. PMID:25648373

  17. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

    SciTech Connect

    Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul

    2008-05-01

    A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.

  18. Genome Sequence of Ureaplasma diversum Strain ATCC 49782

    PubMed Central

    Marques, Lucas M.; Guimarães, Ana M. S.; Martins, Hellen B.; Rezende, Izadora S.; Barbosa, Maysa S.; Campos, Guilherme B.; do Nascimento, Naíla C.; dos Santos, Andrea P.; Amorim, Aline T.; Santos, Verena M.; Messick, Joanne B.

    2015-01-01

    Here, we report the complete genome sequence of Ureaplasma diversum strain ATCC 49782. This species is of bovine origin, having an association with reproductive disorders in cattle, including placentitis, fetal alveolitis, abortion, and birth of weak calves. It has a small circular chromosome of 975,425 bp. PMID:25883297

  19. Genome Sequence of Ureaplasma diversum Strain ATCC 49782.

    PubMed

    Marques, Lucas M; Guimarães, Ana M S; Martins, Hellen B; Rezende, Izadora S; Barbosa, Maysa S; Campos, Guilherme B; do Nascimento, Naíla C; Dos Santos, Andrea P; Amorim, Aline T; Santos, Verena M; Messick, Joanne B; Timenetsky, Jorge

    2015-04-16

    Here, we report the complete genome sequence of Ureaplasma diversum strain ATCC 49782. This species is of bovine origin, having an association with reproductive disorders in cattle, including placentitis, fetal alveolitis, abortion, and birth of weak calves. It has a small circular chromosome of 975,425 bp.

  20. Draft Genome Sequence of Vibrio (Listonella) anguillarum ATCC 14181

    PubMed Central

    Grim, Christopher J.

    2016-01-01

    We report the draft genome sequence of Vibrio anguillarum ATCC 14181, a Gram-negative, hemolytic, O2 serotype marine bacterium that causes mortality in mariculture species. The availability of this genome sequence will add to our knowledge of diversity and virulence mechanisms of Vibrio anguillarum as well as other pathogenic Vibrio spp.

  1. Draft Genome Sequence of Rhodococcus rhodochrous Strain ATCC 21198

    SciTech Connect

    Shields-Menard, Sara A.; Brown, Steven D; Klingeman, Dawn Marie; Indest, Karl; Hancock, Dawn; Wewalwela, Jayani; French, Todd; Donaldson, Janet

    2014-01-01

    Rhodococcus rhodochrous is a Gram-positive red-pigmented bacterium commonly found in the soil. The draft genome sequence for R. rhodochrous strain ATCC 21198 is presented here to provide genetic data for a better understanding of its lipid-accumulating capabilities.

  2. Draft Genome Sequence of Mycobacterium interjectum Strain ATCC 51457T

    PubMed Central

    Levasseur, Anthony; Asmar, Shady; Robert, Catherine

    2016-01-01

    Mycobacterium interjectum is a nontuberculosis species rarely responsible for human infection. The draft genome of M. interjectum ATCC 51457T comprises 5,927,979 bp, exhibiting 67.91% G+C content, 5,314 protein-coding genes, and 51 predicted RNA genes. PMID:27231376

  3. Draft Genome Sequence of Mycobacterium houstonense Strain ATCC 49403T

    PubMed Central

    Levasseur, Anthony; Asmar, Shady; Robert, Catherine

    2016-01-01

    Mycobacterium houstonense is a nontuberculous species rarely responsible for human infection. The draft genome of M. houstonense ATCC 49403T comprises 6,451,020 bp, exhibiting a 66.96% G+C content, 5,881 protein-coding genes, and 65 predicted RNA genes. PMID:27231371

  4. Draft Genome Sequence of Bacillus megaterium Type Strain ATCC 14581

    PubMed Central

    Arya, Gitanjali; Petronella, Nicholas; Crosthwait, Jennifer; Carrillo, Catherine D.

    2014-01-01

    Bacillus megaterium is a Gram-positive, rod-shaped, spore-forming bacterium of biotechnological importance. Here, we report a 5.7-Mbp draft genome sequence of B. megaterium ATCC 14581, which is the type strain of the species. PMID:25395629

  5. Draft Genome Sequence of Bacillus megaterium Type Strain ATCC 14581.

    PubMed

    Arya, Gitanjali; Petronella, Nicholas; Crosthwait, Jennifer; Carrillo, Catherine D; Shwed, Philip S

    2014-01-01

    Bacillus megaterium is a Gram-positive, rod-shaped, spore-forming bacterium of biotechnological importance. Here, we report a 5.7-Mbp draft genome sequence of B. megaterium ATCC 14581, which is the type strain of the species. PMID:25395629

  6. Complete genome sequence of Campylobacter gracilis ATCC 33236T

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The human oral pathogen Campylobacter gracilis has been isolated from periodontal and endodontal infections, and also from non-oral head, neck or lung infections. This study describes the whole-genome sequence of the human periodontal isolate ATCC 33236T (=FDC 1084), which is the first closed genome...

  7. Draft Genome Sequence of Rhizobium rhizogenes Strain ATCC 15834

    PubMed Central

    Kajala, Kaisa; Coil, David A.

    2014-01-01

    Here, we present the draft genome of Rhizobium rhizogenes strain ATCC 15834. The genome contains 7,070,307 bp in 43 scaffolds. R. rhizogenes, also known as Agrobacterium rhizogenes, is a plant pathogen that causes hairy root disease. This hairy root induction has been used in biotechnology for the generation of transgenic root cultures. PMID:25359916

  8. Draft Genome Sequence of Alicyclobacillus acidoterrestris Strain ATCC 49025

    PubMed Central

    Pasvolsky, Ronit; Sela, Noa; Green, Stefan J.; Zakin, Varda

    2013-01-01

    Alicyclobacillus acidoterrestris is a spore-forming Gram-positive, thermo-acidophilic, nonpathogenic bacterium which contaminates commercial pasteurized fruit juices. The draft genome sequence for A. acidoterrestris strain ATCC 49025 is reported here, providing genetic data relevant to the successful adaptation and survival of this strain in its ecological niche. PMID:24009113

  9. Draft Genome Sequence of Strain ATCC 33958, Reported To Be Elizabethkingia miricola

    PubMed Central

    Matyi, Stephanie A.; Hoyt, Peter R.; Ayoubi-Canaan, Patricia; Hasan, Nabeeh A.

    2015-01-01

    We report the draft genome of Elizabethkingia strain ATCC 33958, which has been classified as Elizabethkingia miricola. Similar to other Elizabethkingia species, the ATCC 33958 draft genome contains numerous β-lactamase genes. ATCC 33958 also harbors a urease gene cluster which supports classification as E. miricola. PMID:26205869

  10. 40 CFR 180.1205 - Beauveria bassiana ATCC #74040; exemption from the requirements of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Beauveria bassiana ATCC #74040... RESIDUES IN FOOD Exemptions From Tolerances § 180.1205 Beauveria bassiana ATCC #74040; exemption from the... the insecticide Beauveria bassiana (ATCC #74040) in or on all food commodities when applied or used...

  11. 40 CFR 180.1205 - Beauveria bassiana ATCC #74040; exemption from the requirements of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Beauveria bassiana ATCC #74040... RESIDUES IN FOOD Exemptions From Tolerances § 180.1205 Beauveria bassiana ATCC #74040; exemption from the... the insecticide Beauveria bassiana (ATCC #74040) in or on all food commodities when applied or used...

  12. Spectrophotometric evaluation of selenium binding by Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 yeast.

    PubMed

    Kieliszek, Marek; Błażejak, Stanisław; Płaczek, Maciej

    2016-05-01

    In this study, the ability of selenium binding the biomas of Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 was investigated. Sodium selenite(IV) salts were added to the experimental media at concentrations of 10, 20, 40, and 60 mg Se(4+) L(-1). In the tested concentration range, one concentration reported a significant reduction in the biomass yield of both yeast strains. Intense growth was observed for C. utilis yeast, which reached the highest biomass yield of 15 gd.w.L(-1) after 24h cultivation in the presence of 10mg Se(4+) L(-1). Based on the use of spectrophotometric method for the determination of selenium content by using Variamine Blue as a chromogenic agent, efficient accumulation of this element in the biomass of the investigated yeast was observed. The highest amount of selenium, that is, 5.64 mg Se(4+)gd.w.(-1), was bound from the environment by S. cerevisiae ATCC MYA-2200 cultured in the presence of 60 mg Se(4+) L(-1) medium 72h Slightly less amount, 5.47 mg Se(4+) gd.w.(-1), was absorbed by C. utilis ATCC 9950 during similar cultural conditions. Based on the results of the biomass yield and the use of selenium from the medium, it can be observed that yeasts of the genus Candida are more efficient in binding this element, and this property finds practical application in the production of selenium-enriched yeast.

  13. Spectrophotometric evaluation of selenium binding by Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 yeast.

    PubMed

    Kieliszek, Marek; Błażejak, Stanisław; Płaczek, Maciej

    2016-05-01

    In this study, the ability of selenium binding the biomas of Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 was investigated. Sodium selenite(IV) salts were added to the experimental media at concentrations of 10, 20, 40, and 60 mg Se(4+) L(-1). In the tested concentration range, one concentration reported a significant reduction in the biomass yield of both yeast strains. Intense growth was observed for C. utilis yeast, which reached the highest biomass yield of 15 gd.w.L(-1) after 24h cultivation in the presence of 10mg Se(4+) L(-1). Based on the use of spectrophotometric method for the determination of selenium content by using Variamine Blue as a chromogenic agent, efficient accumulation of this element in the biomass of the investigated yeast was observed. The highest amount of selenium, that is, 5.64 mg Se(4+)gd.w.(-1), was bound from the environment by S. cerevisiae ATCC MYA-2200 cultured in the presence of 60 mg Se(4+) L(-1) medium 72h Slightly less amount, 5.47 mg Se(4+) gd.w.(-1), was absorbed by C. utilis ATCC 9950 during similar cultural conditions. Based on the results of the biomass yield and the use of selenium from the medium, it can be observed that yeasts of the genus Candida are more efficient in binding this element, and this property finds practical application in the production of selenium-enriched yeast. PMID:27049131

  14. Actinobacillus actinomycetemcomitans in Brazilian insulin-dependent individuals with diabetes mellitus.

    PubMed

    Feitosa, A C; de Uzeda, M; Novaes, A B

    1992-01-01

    The prevalence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque specimens from 26 insulin-dependent diabetes mellitus patients, 11-25 years of age, was determined between January 1987 and December 1989. One hundred and thirty subgingival plaque samples were collected with sterile periodontal curettes. The specimens were weighted, diluted, inoculated on trypticase-soy-serum-bacitracin-vancomycin agar medium (TSBV) and incubated under microacrophilic conditions. Aa was isolated from 2.3% of healthy periodontal areas in these patients, while the microorganism was found in 12.5% of the sites with gingivitis and in 2.6% of the periodontal pockets examined. Although biochemical tests used for the characterization of Aa strains showed homogeneous results, different biotypes were isolated from one or more periodontal sites in the same patient.

  15. Electron microscopic examination of capsular material from various serotypes of Actinobacillus pleuropneumoniae.

    PubMed Central

    Jacques, M; Foiry, B; Higgins, R; Mittal, K R

    1988-01-01

    The capsular material on PPLO broth-grown cells of Actinobacillus pleuropneumoniae representing serotypes 1 to 10 was visualized by transmission electron microscopy after polycationic ferritin labeling and also after stabilization with specific antibodies. All the isolates examined were covered with a layer of capsular material whose thickness varied between 80 to 90 nm and 210 to 230 nm when examined by immunostabilization. We were also able to visualize A. pleuropneumoniae in lungs of infected pigs and to estimate the amount of capsular material covering the cells. Our results indicate that differences in capsular structure exist among the different A. pleuropneumoniae serotypes, and this result may explain in part why the serotypes are not equally virulent. Images PMID:3384814

  16. Characterization of the lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4 and N27.

    PubMed Central

    Kiley, P; Holt, S C

    1980-01-01

    The lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans strains Y4 and N27 was isolated by the phenol-water procedure. Morphologically, the molecule consisted of ribbon and branched filaments which comprised 3% of the cellular dry weight. Chemical analysis of the isolated and purified LPSs of both strains showed them to consist of carbohydrate, lipid, 2-keto-3-deoxyoctonate, heptose, hexosamine, and phosphate. The major fatty acids of the lipid A moiety were saturated C14 and beta-OH C14 compounds. Rhamnose, fucose, galactose, glucose, heptose, glucosamine, and galactosamine comprised the monosaccharide portion of the LPS. Biological activity studies revealed both LPS molecules to be active in the Schwartzman reaction and in in vitro 45Ca bone resorption, as well as in macrophage activation and lethality and in platelet aggregation. Images Fig. 1 Fig. 2 Fig. 4 PMID:7228391

  17. Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test

    PubMed Central

    MORIOKA, Ayako; SHIMAZAKI, Yoko; UCHIYAMA, Mariko; SUZUKI, Shoko

    2016-01-01

    We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2. PMID:26726101

  18. Isolation of Actinobacillus seminis from a goat with clinical epididymo-orchitis in Brazil

    PubMed Central

    dos Santos, Fabrine Alexandre; de Azevedo, Edísio Oliveira; de Azevedo, Sérgio Santos; Júnior, Felício Garino; Mota, Rinaldo Aparecido; de Cássia Peixoto Kim, Pomy; Gomes, Ana Lisa Vale; Alves, Clebert José

    2014-01-01

    The present study reports the first isolation of Actinobacillus seminis from a goat in Brazil. A four-year-old Moxotó breeding goat in a flock of 70 goats and 65 sheep reared together in the county of Patos, semiarid region of Northeastern Brazil, showed clinical signs of unilateral orchitis and epididymitis. Diagnosis of A. seminis infection was confirmed by association of clinical findings, bacterial isolation and 16S rRNA gene sequencing. This result suggests that A. seminis may be an additional cause of infertility in goats, and that sheep may be the source of infection because the mixed farming system allows the contact between sheep and goats in the semiarid region of Northeastern Brazil. PMID:24948932

  19. Use of 16S rRNA Sequencing for Identification of Actinobacillus ureae Isolated from a Cerebrospinal Fluid Sample

    PubMed Central

    Whitelaw, A. C.; Shankland, I. M.; Elisha, B. G.

    2002-01-01

    Actinobacillus ureae, previously Pasteurella ureae, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to A. ureae. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene. PMID:11825992

  20. Iron-Chelating Activity of Tetracyclines and Its Impact on the Susceptibility of Actinobacillus actinomycetemcomitans to These Antibiotics

    PubMed Central

    Grenier, Daniel; Huot, Marie-Pierre; Mayrand, Denis

    2000-01-01

    Three tetracyclines (tetracycline, doxycycline, and minocycline) were found to possess iron-chelating activity in a colorimetric siderophore assay. Determination of MICs indicated that the activity of doxycycline against the periodontopathogen Actinobacillus actinomycetemcomitans was only slightly influenced by the presence of an excess of iron that likely saturates the antibiotic. On the other hand, the MICs of doxycycline and minocycline were significantly lower for A. actinomycetemcomitans cultivated under iron-poor conditions than under iron-rich conditions. PMID:10681353

  1. Draft Genome Sequence of Mycobacterium chelonae Type Strain ATCC 35752

    PubMed Central

    Hasan, Nabeeh A.; Davidson, Rebecca M.; de Moura, Vinicius Calado Nogueira; Garcia, Benjamin J.; Reynolds, Paul R.; Epperson, L. Elaine; Farias-Hesson, Eveline; DeGroote, Mary Ann; Jackson, Mary

    2015-01-01

    Mycobacterium chelonae is a rapidly growing opportunistic nontuberculous mycobacterial (NTM) species that causes infections in humans and other hosts. Here, we report the draft genome sequence of Mycobacterium chelonae type strain ATCC 35752, consisting of 4.89 Mbp, 63.96% G+C content, 4,489 protein-coding genes, 48 tRNAs, and 3 rRNA genes. PMID:26021923

  2. Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

    DOEpatents

    Adney, W.S.; Thomas, S.R.; Nieves, R.A.; Himmel, M.E.

    1994-11-22

    A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C[sub 1], and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81 C at pH's from about 2 to about 9, and at a inactivation temperature of about 100 C at pH's from about 2 to about 9. 9 figs.

  3. Magnetic response in cultures of Streptococcus mutans ATCC-27607.

    PubMed

    Adamkiewicz, V W; Bassous, C; Morency, D; Lorrain, P; Lepage, J L

    1987-01-01

    Streptococcus mutans ATCC-27607 produces exopolysaccharides that adhere to glass. In the normal geomagnetic field about 50% more polysaccharide adhere preferentially to glass surfaces facing North as compared to South facing surfaces. Reversal of the direction of the magnetic field by 180 degrees produces a similar reversal in the direction of the preferential accumulation. Reduction of the field by 90% abolishes the preferential accumulation. PMID:3582582

  4. Cellulose produced by Gluconacetobacter xylinus strains ATCC 53524 and ATCC 23768: Pellicle formation, post-synthesis aggregation and fiber density.

    PubMed

    Lee, Christopher M; Gu, Jin; Kafle, Kabindra; Catchmark, Jeffrey; Kim, Seong H

    2015-11-20

    The pellicle formation, crystallinity, and bundling of cellulose microfibrils produced by bacterium Gluconacetobacter xylinus were studied. Cellulose pellicles were produced by two strains (ATCC 53524 and ATCC 23769) for 1 and 7 days; pellicles were analyzed with scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrational sum-frequency-generation (SFG) spectroscopy, and attenuated total reflectance infrared (ATR-IR) spectroscopy. The bacterial cell population was higher at the surface exposed to air, indicating that the newly synthesized cellulose is deposited at the top of the pellicle. XRD, ATR-IR, and SFG analyses found no significant changes in the cellulose crystallinity, crystal size or polymorphic distribution with the culture time. However, SEM and SFG analyses revealed cellulose macrofibrils produced for 7 days had a higher packing density at the top of the pellicle, compared to the bottom. These findings suggest that the physical properties of cellulose microfibrils are different locally within the bacterial pellicles. PMID:26344281

  5. Cellulose produced by Gluconacetobacter xylinus strains ATCC 53524 and ATCC 23768: Pellicle formation, post-synthesis aggregation and fiber density.

    PubMed

    Lee, Christopher M; Gu, Jin; Kafle, Kabindra; Catchmark, Jeffrey; Kim, Seong H

    2015-11-20

    The pellicle formation, crystallinity, and bundling of cellulose microfibrils produced by bacterium Gluconacetobacter xylinus were studied. Cellulose pellicles were produced by two strains (ATCC 53524 and ATCC 23769) for 1 and 7 days; pellicles were analyzed with scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrational sum-frequency-generation (SFG) spectroscopy, and attenuated total reflectance infrared (ATR-IR) spectroscopy. The bacterial cell population was higher at the surface exposed to air, indicating that the newly synthesized cellulose is deposited at the top of the pellicle. XRD, ATR-IR, and SFG analyses found no significant changes in the cellulose crystallinity, crystal size or polymorphic distribution with the culture time. However, SEM and SFG analyses revealed cellulose macrofibrils produced for 7 days had a higher packing density at the top of the pellicle, compared to the bottom. These findings suggest that the physical properties of cellulose microfibrils are different locally within the bacterial pellicles.

  6. Pharmacokinetics of tildipirosin in porcine plasma, lung tissue, and bronchial fluid and effects of test conditions on in vitro activity against reference strains and field isolates of Actinobacillus pleuropneumoniae.

    PubMed

    Rose, M; Menge, M; Bohland, C; Zschiesche, E; Wilhelm, C; Kilp, S; Metz, W; Allan, M; Röpke, R; Nürnberger, M

    2013-04-01

    The pharmacokinetics of tildipirosin (Zuprevo(®) 40 mg/mL solution for injection for pigs), a novel 16-membered-ring macrolide for the treatment for swine respiratory disease (SRD), was investigated in studies collecting blood plasma and postmortem samples of lung tissue and bronchial fluid (BF) from swine. In view of factors influencing the in vitro activity of macrolides, and for the interpretation of tildipirosin pharmacokinetics in relation to minimum inhibitory concentrations (MIC), additional experiments were conducted to study the effects of pH, carbon dioxide-enriched atmosphere, buffers, and serum on tildipirosin MICs for various reference strains and Actinobacillus (A.) pleuropneumoniae field isolates. After single intramuscular (i.m.) injection at 4 mg/kg body weight, maximum plasma concentration (Cmax) was 0.9 μg/mL observed within 23 min (Tmax ). Mean residence time from the time of dosing to the time of last measurable concentration (MRTlast) and terminal half-life (T1/2) both were about 4 days. A dose-response relationship with no significant sex effect is observed for area under the plasma concentration-time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUClast) over the range of doses up to 6 mg/kg. However, linear dose proportionality could not be proven with statistical methods. The time-concentration profile of tildipirosin in BF and lung far exceeded that in blood plasma. In lung, tildipirosin concentrations reached 3.1 μg/g at 2 h, peaked at 4.3 μg/g at day 1, and slowly declined to 0.8 μg/g at day 17. In BF, tildipirosin levels were 14.3, 7.0, and 6.5 μg/g at days 5, 10, and 14. T1/2 in lung was ∼7 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination. Culture media pH and carbon dioxide-enriched atmosphere (CO2 -EA) had a marked impact on in vitro activity of tildipirosin in reference strains of various rapidly growing aerobic and

  7. Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, Michael E.; Adney, William S.; Tucker, Melvin P.; Grohmann, Karel

    1994-01-01

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068). The cellulase is water soluble, possesses both C.sub.1 and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83.degree. C. at pH's from about 2 to about 9, and in inactivation temperature of about 110.degree. C. at pH's from about 2 to about 9.

  8. Draft genome sequence of Rhodococcus rhodochrous strain ATCC 17895

    PubMed Central

    Chen, Bi-Shuang; Otten, Linda G.; Resch, Verena; Muyzer, Gerard; Hanefeld, Ulf

    2013-01-01

    Rhodococcus rhodochrous ATCC 17895 possesses an array of mono- and dioxygenases, as well as hydratases, which makes it an interesting organism for biocatalysis. R. rhodochrous is a Gram-positive aerobic bacterium with a rod-like morphology. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Based on small subunit rRNA analysis, the strain is more likely to be a strain of Rhodococcus erythropolis rather than Rhodococcus rhodochrous. PMID:24501654

  9. Thermostable purified endoglucanase from Acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, M.E.; Adney, W.S.; Tucker, M.P.; Grohmann, K.

    1994-01-04

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068) is presented. The cellulase is water soluble, possesses both C[sub 1] and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83 C at pH's from about 2 to about 9, and in inactivation temperature of about 110 C at pH's from about 2 to about 9. 7 figures.

  10. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  11. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  12. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  13. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  14. Expression and phylogenetic relationships of a novel lacZ homologue from Actinobacillus pleuropneumoniae.

    PubMed

    Anderson, T J; MacInnes, J I

    1997-07-01

    To learn more about the genetics and physiology of the important swine pathogen, Actinobacillus pleuropneumoniae, we cloned the lacZ gene by complementation of an Escherichia coli delta lac mutant. The A. pleuropneumoniae lacZ gene has an open reading frame of 3015 bp which could encode a protein with a predicted molecular mass of 117022. The deduced protein shares 26.8-34.8% identity with beta-galactosidases from both Gram-positive and Gram-negative bacteria. Sequences with homology to seven regions commonly found in beta-galactosidases are present and amino acids corresponding to active site residues Tyr-503 and Glu-537 in E. coli LacZ are also conserved; however, there is a leucine in the place of Gly-794, a residue which has been implicated in substrate recognition. The sequences flanking the A. pleuropneumoniae lacZ gene do not share homology with known transport or regulatory genes nor do they share homology with cAMP receptor protein (CRP) or LacI binding sites. Low levels of beta-galactosidase activity could be detected when the protein was expressed from a multicopy plasmid in E. coli delta lac and when it was measured in A. pleuropneumoniae. The level of activity was not markedly reduced in the presence of glucose. Although the A. pleuropneumoniae LacZ shares some features with other beta-galactosidases, its constitutive expression and an unusual active site residue suggest that it may have a unique function. PMID:9228778

  15. Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae.

    PubMed Central

    Frey, J; Perrin, J; Nicolet, J

    1989-01-01

    The genetic determinant of the cohemolysin which is responsible for the CAMP phenomenon, a cohemolysis, of Actinobacillus pleuropneumoniae was cloned in Escherichia coli. Total DNA from the A. pleuropneumoniae serotype 1 type strain 4074 was used to construct a gene library in plasmid pUC18 in E. coli JM83. A total of 10,500 clones containing recombinant plasmids have been screened for hemolysis on blood plates. Fifty-five clones which showed a weak hemolytic response after 24 to 48 h of incubation were screened for the CAMP reaction with Staphylococcus aureus. This led to the identification of one clone which showed a positive CAMP reaction. Immunoblot analysis revealed that the recombinant strain expressed a protein with a molecular mass of 27,000 daltons, similar in size to the CAMP protein of the group B streptococci. Rabbit antibodies against the CAMP+ clone neutralized the CAMP reaction mediated by the E. coli strain containing the cloned CAMP gene as well as that of A. pleuropneumoniae. Antibodies raised against the cloned CAMP cohemolysin cross-reacted with Streptococcus agalactiae protein B. We designate the 27,000-dalton molecule CAMP factor protein and name its corresponding gene cfp. Images PMID:2659534

  16. Preparation, characterization, and immunogenicity of conjugate vaccines directed against Actinobacillus pleuropneumoniae virulence determinants.

    PubMed Central

    Byrd, W; Kadis, S

    1992-01-01

    Conjugate vaccines were prepared in an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP). Two subunit conjugates were prepared by coupling the A. pleuropneumoniae 4074 serotype 1 capsular polysaccharide (CP) to the hemolysin protein (HP) and the lipopolysaccharide (LPS) to the HP. Adipic acid dihydrazide was used as a spacer to facilitate the conjugation in a carbodiimide-mediated reaction. The CP and the LPS were found to be covalently coupled to the HP in the conjugates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detergent gel chromatography analyses. Following a booster vaccination, pigs exhibited significantly high (P less than 0.05) immunoglobulin G antibodies against CP, LPS, and HP. The anti-CP and anti-LPS immunoglobulin G antibodies were found to function as opsonins in the phagocytosis of A. pleuropneumoniae by polymorphonuclear leukocytes, whereas antibodies to the HP neutralized the cytotoxic effect of the HP on polymorphonuclear leukocytes. No killing of A. pleuropneumoniae was observed when the effects of the antibodies were tested in the presence of complement. Thus, polysaccharide-protein A. pleuropneumoniae conjugates elicit significant antibody responses against each component of each conjugate, which could be instrumental in protecting swine against SPAP. Images PMID:1639471

  17. Purification and characterization of the serotype c antigen from Actinobacillus actinomycetemcomitans.

    PubMed Central

    Zambon, J J; Slots, J; Miyasaki, K; Linzer, R; Cohen, R; Levine, M; Genco, R J

    1984-01-01

    The serotype c antigen from Actinobacillus actinomycetemcomitans was purified with fractional ethanol precipitation of cell-free culture supernatant, sequential ion-exchange chromatography, and gel filtration chromatography. The preparation obtained demonstrated a single precipitin line in immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis when rabbit antisera to serotype c whole bacterial cells were used. No immunological reaction was detected with antisera to serotype c lipopolysaccharide, indicating that lipopolysaccharide was not present in the preparation. The serotype c antigen was composed of 95% carbohydrate, 2% protein, and 3.1% phosphate. Gas chromatographic analysis of the antigen obtained from growth in either complex or chemically defined media revealed that the carbohydrate constituent was composed of 84 to 90.1% mannose, 4.8 to 16% glucose, 1.9% N-acetylglucosamine, 1.4% fucose, and 0.2% galactose. The present data suggest that A. actinomycetemcomitans serotype c antigen is predominantly a mannose-containing carbohydrate suggestive of a mannan. Images PMID:6423542

  18. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss. PMID:26817919

  19. Secreted proteases from Actinobacillus pleuropneumoniae serotype 1 degrade porcine gelatin, hemoglobin and immunoglobulin A.

    PubMed Central

    Negrete-Abascal, E; Tenorio, V R; Serrano, J J; Garcia, C; de la Garza, M

    1994-01-01

    It was found that 48 hour cultures of Actinobacillus pleuropneumoniae secreted proteases into the medium. Electrophoresis in polyacrylamide gels (10%) copolymerized with porcine gelatin (0.1%), of the 70% (NH4)2SO4 precipitate from the culture supernatants, displayed protease activities of different molecular weights: > 200, 200, 90, 80, 70 and 50 kDa. They had activity over a broad range of pHs (4-8), with an optimal pH of 6-7. All were inhibited by 10 mM EDTA, and reactivated by 10 mM calcium. They were stable at -20 degrees C for more than a month. The proteases also degraded porcine IgA and porcine, human, and bovine hemoglobin, although they appeared to be less active against the hemoglobins. The IgA was totally cleaved in 48 h, using supernatants concentrated with polyvinyl pyrrolidone or the 70% (NH4)2SO4. Extracellular proteases could play a role in virulence. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:8004545

  20. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss.

  1. In vitro activity of azithromycin compared with that of erythromycin against Actinobacillus actinomycetemcomitans.

    PubMed Central

    Pajukanta, R; Asikainen, S; Saarela, M; Alaluusua, S; Jousimies-Somer, H

    1992-01-01

    The in vitro susceptibility of Actinobacillus actinomycetemcomitans to azithromycin, a new macrolide antibiotic of a new class known as azalides, was compared with that of erythromycin by the agar dilution method on Mueller-Hinton Haemophilus test medium. Eighty-two A. actinomycetemcomitans strains, 79 recent clinical isolates obtained from 40 periodontally healthy or diseased subjects, and 3 type strains were included in the study. Erythromycin showed poor in vitro activity against A. actinomycetemcomitans. Azithromycin, however, was highly effective against A. actinomycetemcomitans: all strains were inhibited at 2.0 micrograms/ml. Azithromycin exhibited the best in vitro activity against the serotype a subpopulation of A. actinomycetemcomitans: 100% of the strains were inhibited at 1.0 micrograms/ml. The lowest MICs were, however, recorded by serotype b strains. Since azithromycin has favorable pharmacokinetic properties, including excellent distribution into tissues, it could be expected to pass into gingival crevicular fluid at levels sufficient to inhibit A. actinomycetemcomitans in vivo. Therefore, it is a good candidate for future clinical trials in A. actinomycetemcomitans-associated periodontitis. PMID:1329617

  2. Clonal analysis of the Actinobacillus pleuropneumoniae population in a geographically restricted area by multilocus enzyme electrophoresis.

    PubMed Central

    Møller, K; Nielsen, R; Andersen, L V; Kilian, M

    1992-01-01

    The genetic diversity among 250 isolates of Actinobacillus pleuropneumoniae from lungs of pigs with pleuropneumonia and from tonsils of apparently healthy pigs at slaughter was estimated by multilocus enzyme electrophoresis. The Danish strains were derived from both specific-pathogen-free and conventional herds. Sixty-six percent of the isolates belonged to three electrophoretic types (ETs) of a total of 37 ETs detected. While five biotype 2 isolates constituted a separate ET closely related to biotype 1 isolates, the type strain of the species (Shope 4074) belonged to its own ET, with a genetic distance of 0.30 from its nearest neighbor. Isolates of serotypes traditionally considered to have less pathogenic potential (serotypes 6, 10, and 12) from herds with acute outbreaks of pleuropneumonia belonged to the same ETs as isolates from apparently healthy pigs, suggesting that factors such as cross immunity and management may lead to divergent clinical results. Isolates from four herds harboring more than one serotype showed distinct profiles between the serotypes, indicating no or only limited chromosomal recombination among clones. Isolates from tonsils belonged to the same ET as isolates from lungs. The same ET was isolated from widely different parts of the world. Evidence from this study indicates that multilocus enzyme electrophoresis may be a valuable tool for the epidemiological analysis of A. pleuropneumoniae. PMID:1551979

  3. Expression cloning of a periodontitis-associated apoptotic effector, cagE homologue, in Actinobacillus actinomycetemcomitans.

    PubMed

    Teng, Yen-Tung A; Hu, Wenqi

    2003-04-18

    To study anti-bacterial immunity and to identify critical bacterial antigens associated with specific periodontal infection, we screened the genomic library of Actinobacillus actinomycetemcomitans, a major Gram(-) anaerobe causing human periodontitis, by expression cloning using disease-associated periodontal CD4(+)T cells derived from HuPBL-engrafted NOD/SCID mice. Here, we report one of the novel genes identified and designated, cagE homologue (in short: cagE) of A. actinomycetemcomitans, which encodes a putative bacterial type IV secretion system with significant homology to Helicobacter pylori CagE and Agrobacterium tumefaciens VirB4. All serum samples from A. actinomycetemcomitans-infected periodontitis patients, but not from the healthy controls, readily recognized CagE by ELISA and Western blot, suggesting its biological and clinical significance. The CagE protein, upon secretion, elicited significant apoptosis on primary human epithelia, endothelia, osteoblasts, and T cells by 4-12h in vitro. Importantly, both cagE(-) mutant strain and N-terminus truncated CagE protein drastically reduced (p<0.001) the induction of apoptosis on human epithelia in vitro. These data strongly suggest that a novel effector protein, CagE in A. actinomycetemcomitans, induces apoptosis of human cells and destructive immunity, thereby it may play an important role in the pathogenesis of A. actinomycetemcomitans-mediated infections. PMID:12684047

  4. Identification of genomic clonal types of Actinobacillus actinomycetemcomitans by restriction endonuclease analysis.

    PubMed Central

    Han, N; Hoover, C I; Winkler, J R; Ng, C Y; Armitage, G C

    1991-01-01

    To evaluate its utility in discriminating different strains, restriction endonuclease analysis was applied to 12 strains of Actinobacillus actinomycetemcomitans (3 serotype a, 5 serotype b, and 4 serotype c strains). DNA isolated from each strain was digested by 12 different restriction endonucleases, and the electrophoretic banding patterns of the resulting DNA fragments were compared. The DNA fragment patterns produced by SalI, XhoI, and XbaI for the 12 A. actinomycetemcomitans strains were simple (less than 30 bands) and allowed us to recognize easily 10 distinct genomic clonal types. The three serotype a strains exhibited distinctly different clonal types from one another, the five serotype b strains exhibited an additional four distinct clonal types, and the four serotype c strains showed another three different clonal types. The other endonucleases tested were less useful in typing A. actinomycetemcomitans. We conclude that restriction endonuclease analysis is a powerful tool for typing and discerning genetic heterogeneity and homogeneity among A. actinomycetemcomitans strains. It should, therefore, be very useful for epidemiologic studies. Images PMID:1761677

  5. Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence.

    PubMed

    Xiao, Longwen; Zhou, Liang; Sun, Changjiang; Feng, Xin; Du, ChongTao; Gao, Yu; Ji, Qun; Yang, Shuxin; Wang, Yu; Han, Wenyu; Langford, P R; Lei, Liancheng

    2012-10-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, and adherence to host cells is a key step in the pathogenic process. Although trimeric autotransporter adhesins (TAAs) were identified in many pathogenic bacteria in recent years, none in A. pleuropneumoniae have been characterized. In this study, we identified a TAA from A. pleuropneumoniae, Apa, and characterized the contribution of its amino acid residues to the adhesion process. Sequence analysis of the C-terminal amino acid residues of Apa revealed the presence of a putative translocator domain and six conserved HsfBD1-like or HsfBD2-like binding domains. Western blot analysis revealed that the 126 C-terminal amino acids of Apa could form trimeric molecules. By confocal laser scanning microscopy, one of these six domains (ApaBD3) was determined to mediate adherence to epithelial cells. Adherence assays and adherence inhibition assays using a recombinant E. coli- ApaBD3 strain which expressed ApaBD3 on the surface of E. coli confirmed that this domain was responsible for the adhesion activity. Moreover, cellular enzyme-linked immunosorbent assays demonstrated that ApaBD3 mediated high-level adherence to epithelial cell lines. Intriguingly, autoagglutination was observed with the E. coli- ApaBD3 strain, and this phenomenon was dependent upon the association of the expressed ApaBD3 with the C-terminal translocator domain.

  6. [Generation of nalidixic acid-resistant strains and signature-tagged mutants of Actinobacillus pleuropneumoniae].

    PubMed

    Shang, Lin; Li, Wei; Li, Liangjun; Li, Lu; Zhang, Sihua; Li, Tingting; Li, Yaokun; Liu, Lei; Guo, Zhiwei; Zhou, Rui; Chen, Huanchun

    2008-01-01

    Actinobacillus pleuropneumoniae is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Signature-tagged mutagenesis (STM) is an effective method to identify virulence genes in bacteria. In this study, we selected nalidixic acid-resistant strains of APP serotypes 1 and 3 by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CC 118 lambdapir or S17-1 lambdapir containing mini-Tn10 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5alpha (pRK2073). We screened mutant strains by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains could easily be induced in vitro and the resistance was due to the mutation in the DNA gyrase A subunit gene gyrA. In the mating experiments, the bi-parental mating was more effective and easier than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data were helpful for the construction of STM mutants and pickup of virulence genes of APP. PMID:18338580

  7. Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae.

    PubMed

    Yang, Wang; Pin, Chen; Haibing, Guo; Yang, Chen; Hui, Liu; Qigai, He

    2009-11-01

    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (chi(2) test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.

  8. Detection and Identification of Actinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

    PubMed Central

    Lo, Terry M.; Ward, Christine K.; Inzana, Thomas J.

    1998-01-01

    Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 102 CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay. PMID:9620404

  9. Immunodominant antigen of Actinobacillus actinomycetemcomitans Y4 in high-responder patients.

    PubMed Central

    Califano, J V; Schenkein, H A; Tew, J G

    1989-01-01

    This study was undertaken to look for characteristics of the immunodominant antigen(s) of Actinobacillus actinomycetemcomitans Y4 that might help explain the high antibody titers in periodontitis patients. Radioimmunoassays (RIA) were performed on sera from 481 patients; sera from the 32 patients with the highest anti-Y4 titers (above 128,000 RIA U/ml) were further analyzed. Y4 antigen was boiled for 45 min or treated with papain, and antibody responses were analyzed by RIA and Western blotting (immunoblotting). In addition, carbohydrate was purified from Y4 and examined by Western blotting. The results indicated that the immunodominant antigen of Y4 in high responders was stable after papain treatment or boiling for 45 min. Papain or boiling eliminated protein bands but a large diffuse band persisted on Western blots. With increasing dilutions of sera, bands on Western blots corresponding to protein antigens disappeared, while the large diffuse band resembling that of carbohydrate persisted. Partially purified Y4 carbohydrate contained the large diffuse band. Double-immunodiffusion analysis indicated that rabbit serotype b-specific antiserum and patient sera recognized the same antigen. When the carbohydrate extract was passed over a lipid A-binding column to remove lipopolysaccharide, the smear corresponding to the immunodominant antigen was still present on Western blots. The immunodominant antigen of Y4 in high-responder individuals appears to be a carbohydrate and is possibly the capsular polysaccharide. Images PMID:2496034

  10. Actinobacillus actinomycetemcomitans toxin induces both cell cycle arrest in the G2/M phase and apoptosis.

    PubMed

    Ohguchi, M; Ishisaki, A; Okahashi, N; Koide, M; Koseki, T; Yamato, K; Noguchi, T; Nishihara, T

    1998-12-01

    We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue. PMID:9826381

  11. Preparation, characterization, and immunogenicity of conjugate vaccines directed against Actinobacillus pleuropneumoniae virulence determinants.

    PubMed

    Byrd, W; Kadis, S

    1992-08-01

    Conjugate vaccines were prepared in an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP). Two subunit conjugates were prepared by coupling the A. pleuropneumoniae 4074 serotype 1 capsular polysaccharide (CP) to the hemolysin protein (HP) and the lipopolysaccharide (LPS) to the HP. Adipic acid dihydrazide was used as a spacer to facilitate the conjugation in a carbodiimide-mediated reaction. The CP and the LPS were found to be covalently coupled to the HP in the conjugates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detergent gel chromatography analyses. Following a booster vaccination, pigs exhibited significantly high (P less than 0.05) immunoglobulin G antibodies against CP, LPS, and HP. The anti-CP and anti-LPS immunoglobulin G antibodies were found to function as opsonins in the phagocytosis of A. pleuropneumoniae by polymorphonuclear leukocytes, whereas antibodies to the HP neutralized the cytotoxic effect of the HP on polymorphonuclear leukocytes. No killing of A. pleuropneumoniae was observed when the effects of the antibodies were tested in the presence of complement. Thus, polysaccharide-protein A. pleuropneumoniae conjugates elicit significant antibody responses against each component of each conjugate, which could be instrumental in protecting swine against SPAP. PMID:1639471

  12. Efficacy of tilmicosin in the control of experimentally induced Actinobacillus pleuropneumoniae infection in swine.

    PubMed

    Paradis, Marie-Anne; Vessie, Gordon H; Merrill, John K; Dick, C Paul; Moore, Camille; Charbonneau, George; Gottschalk, M; MacInnes, Janet I; Higgins, Robert; Mittal, K R; Girard, C; Aramini, Jeffery J; Wilson, Jeffrey B

    2004-01-01

    The efficacy of tilmicosin administered in the feed to control Actinobacillus pleuropneumoniae infections in pigs was evaluated through a multisite, multitrial study. For each of 6 trials, 48 pigs (stratified by weight and sex) were randomly assigned to 6 to 8 pens. Medicated feed containing tilmicosin (200 g/t) and unmedicated feed were randomly assigned at the pen level and were provided ad libitum from day -7 to trial termination (day 14). Seeder pigs (inoculated intranasally with A. pleuropneumoniae serotype 1 and showing signs of clinical disease) were introduced to each pen on day 0. Rates of death, gross lesions, and culture of A. pleuropneumoniae at necropsy, clinical scores, average daily gain in weight, and average body temperature were compared between the medicated and unmedicated pigs. Compared with the unmedicated pigs, significantly fewer (P < 0.05) pigs given tilmicosin had lesions typical of A. pleuropneumoniae or had A. pleuropneumoniae isolated from their tissues at necropsy. Together with a significant reduction (P < 0.05) in the average percentage of pneumonic lung involvement (both visually and by weight), there were reductions in the numbers of pigs with moderate and severe pneumonic lung lesions and with A. pleuropneumoniae associated mortality. With tilmicosin treatment, the average daily weight gain, daily temperature, abdominal appearance, attitude, and respiration were also significantly better (P < 0.05). The results of this study demonstrate the in vivo effectiveness of tilmicosin (200 g/t) in controlling pleuropneumonia among swine experimentally infected with A. pleuropneumoniae.

  13. Assessment of the efficacy of tilmicosin phosphate to eliminate Actinobacillus pleuropneumoniae from carrier pigs.

    PubMed

    Fittipaldi, N; Klopfenstein, C; Gottschalk, M; Broes, A; Paradis, M A; Dick, C P

    2005-04-01

    The aim of this study was to evaluate the efficacy of in-feed medication with tilmicosin phosphate in order to eliminate or reduce the carriage of Actinobacillus pleuropneumoniae in the tonsils of carrier pigs. Two groups of 6 carrier animals received either a non-medicated feed (control group) or feed medicated with 400 ppm of tilmicosin phosphate (treated group) for 30 d. Three sentinel pigs were then introduced in each group and left for 29 d. The presence of A. pleuropneumoniae in tonsils was monitored using several techniques, including polymerase chain reaction (PCR). At the end of the treatment all of the control animals, but only 1 treated pig, were positive by PCR from tonsillar surface material. However, at necropsy, all control and most treated animals, as well as 1 sentinel animal, in both groups were positive by PCR from whole tonsils. In conclusion, under the experimental conditions, in-feed treatment with 400 ppm of tilmicosin phosphate significantly reduced the presence of A. pleuropneumoniae on the surface of tonsils but was unable to completely eliminate the organism from deeper tonsillar tissues and to prevent bacterial shedding by carrier animals.

  14. Identification of QTL affecting resistance/susceptibility to acute Actinobacillus pleuropneumoniae infection in swine.

    PubMed

    Reiner, Gerald; Bertsch, Natalie; Hoeltig, Doris; Selke, Martin; Willems, Hermann; Gerlach, Gerald Friedrich; Tuemmler, Burkhard; Probst, Inga; Herwig, Ralf; Drungowski, Mario; Waldmann, Karl Heinz

    2014-04-01

    Actinobacillus pleuropneumoniae is among the most important pathogens worldwide in pig production. The agent can cause severe economic losses due to decreased performance, acute or chronic pleuropneumonia and an increased incidence of death. Therapeutics cannot be used in a sustainable manner, and vaccination is not always available, but discovering more about host defence and disease mechanisms might lead to new methods of prophylaxis. The aim of the present study was to detect quantitative trait loci (QTL) associated with resistance/susceptibility to A. pleuropneumoniae. Under controlled conditions, 170 F2 animals of a Hampshire/Landrace family, with known differences in founder populations regarding A. pleuropneumoniae resistance, were challenged with an A. pleuropneumoniae serotype 7 aerosol followed by a detailed clinical, radiographic, ultrasonographic, pathological and bacteriological examination. F2 pigs were genotyped with 159 microsatellite markers. Significant QTL were identified on Sus scrofa chromosomes (SSC) 2, 6, 12, 13, 16, 17 and 18. They explained 6-22% of phenotypic variance. One QTL on SSC2 reached significance on a genome-wide level for five associated phenotypic traits. A multiple regression analysis revealed a combinatory effect of markers SWR345 (SSC2) and S0143 (SSC12) on Respiratory Health Score, Clinical Score and the occurrence of death. The results indicate the genetic background of A. pleuropneumoniae resistance in swine and provide new insights into the genetic architecture of resistance/susceptibility to porcine pleuropneumonia. The results will be helpful in identifying the underlying genes and mechanisms.

  15. Specific Humoral Immune Response Induced by Propionibacterium acnes Can Prevent Actinobacillus pleuropneumoniae Infection in Mice

    PubMed Central

    Yang, Feng; Ma, Qiuyue; Huang, Jing; Ji, Qun; Zhai, Ruidong; Wang, Lei; Wang, Yu; Li, Linxi; Sun, Changjiang; Feng, Xin; Han, Wenyu

    2014-01-01

    Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, has a major impact on economics, ecology, and animal welfare in the pig-rearing industry. Propionibacterium acnes, a facultative anaerobic Gram-positive corynebacterium, exists widely in normal healthy adult animals. We have shown previously that P. acnes can prevent A. pleuropneumoniae infections in mice and pigs. To elucidate the mechanism of this effect and to identify novel A. pleuropneumoniae vaccines, the role of anti-P. acnes antibodies in preventing infection was analyzed by indirect immunofluorescence and opsonophagocytosis assays in vitro. The role of the specific humoral immune response induced by P. acnes was confirmed in a B cell depletion mouse model. The survival rates of mice challenged with A. pleuropneumoniae exhibited a highly significant positive rank correlation with the levels of anti-P. acnes antibodies. The specific antibodies induced by P. acnes had the ability to combine with A. pleuropneumoniae and increase opsonization of A. pleuropneumoniae for phagocytosis. Furthermore, analysis in the murine B cell depletion model confirmed that the humoral immune response induced by P. acnes played an important role in resistance to A. pleuropneumoniae infection. In this study, we further elucidated the reasons that P. acnes can prevent A. pleuropneumoniae infection, which provides useful evidence for the development of heterologous vaccines for the control of porcine contagious pleuropneumonia. PMID:24429068

  16. Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

    PubMed Central

    2006-01-01

    Abstract Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis. PMID:16548331

  17. Production of Biohydrogen from Wastewater by Klebsiella oxytoca ATCC 13182.

    PubMed

    Thakur, Veena; Tiwari, K L; Jadhav, S K

    2015-08-01

    Production of biohydrogen from distillery effluent was carried out by using Klebsiella oxytoca ATCC 13182. The work focuses on optimization of pH, temperature, and state of bacteria, which are the various affecting factors for fermentative biohydrogen production. Results indicates that at 35 °C for suspended cultures, the production was at its maximum (i.e., 91.33 ± 0.88 mL) when compared with other temperatures. At 35 °C and at pH 5 and 6, maximum productions of 117.67 ± 1.45 and 111.67 ± 2.72 mL were observed with no significant difference. When immobilized, Klebsiella oxytoca ATCC 13182 was used for biohydrogen production at optimized conditions, production was 186.33 ± 3.17 mL. Hence, immobilized cells were found to be more advantageous for biological hydrogen production over suspended form. Physicochemical analysis of the effluent was conducted before and after fermentation and the values suggested that the fermentative process is an efficient method for biological treatment of wastewater. PMID:26237683

  18. Production of Biohydrogen from Wastewater by Klebsiella oxytoca ATCC 13182.

    PubMed

    Thakur, Veena; Tiwari, K L; Jadhav, S K

    2015-08-01

    Production of biohydrogen from distillery effluent was carried out by using Klebsiella oxytoca ATCC 13182. The work focuses on optimization of pH, temperature, and state of bacteria, which are the various affecting factors for fermentative biohydrogen production. Results indicates that at 35 °C for suspended cultures, the production was at its maximum (i.e., 91.33 ± 0.88 mL) when compared with other temperatures. At 35 °C and at pH 5 and 6, maximum productions of 117.67 ± 1.45 and 111.67 ± 2.72 mL were observed with no significant difference. When immobilized, Klebsiella oxytoca ATCC 13182 was used for biohydrogen production at optimized conditions, production was 186.33 ± 3.17 mL. Hence, immobilized cells were found to be more advantageous for biological hydrogen production over suspended form. Physicochemical analysis of the effluent was conducted before and after fermentation and the values suggested that the fermentative process is an efficient method for biological treatment of wastewater.

  19. Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212

    PubMed Central

    León-Calvijo, María A.; Leal-Castro, Aura L.; Almanzar-Reina, Giovanni A.; Rosas-Pérez, Jaiver E.; García-Castañeda, Javier E.; Rivera-Monroy, Zuly J.

    2015-01-01

    Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4–33 μM) and E. faecalis (MIC 10–33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield. PMID:25815317

  20. Pseudomonas oleovorans subsp. lubricantis subsp. nov., and reclassification of Pseudomonas pseudoalcaligenes ATCC 17440T as later synonym of Pseudomonas oleovorans ATCC 8062 T.

    PubMed

    Saha, Ratul; Spröer, Cathrin; Beck, Brian; Bagley, Susan

    2010-04-01

    Isolate RS1(T) isolated from used metalworking fluid was found to be a Gram-negative, motile, and non-spore forming rod. Based on phylogenetic analyses with 16S rRNA, isolate RS1(T) was placed into the mendocina sublineage of Pseudomonas. The major whole cell fatty acids were C(18:1)omega7c (32.6%), C(16:0) (25.5%), and C(15:0) ISO 2OH/C(16:1)omega7c (14.4%). The sequence similarities of isolate RS1(T) based on gyrB and rpoD genes were 98.9 and 98.0% with Pseudomonas pseudoalcaligenes, and 98.5 and 98.1% with Pseudomonas oleovorans, respectively. The ribotyping pattern showed a 0.60 similarity with P. oleovorans ATCC 8062(T) and 0.63 with P. pseudoalcaligenes ATCC17440(T). The DNA G + C content of isolate RS1(T) was 62.2 mol.%. The DNA-DNA relatedness was 73.0% with P. oleovorans ATCC 8062(T) and 79.1% with P. pseudoalcaligenes ATCC 17440(T). On the basis of morphological, biochemical, and molecular studies, isolate RS1(T) is considered to represent a new subspecies of P. oleovorans. Furthermore, based on the DNA-DNA relatedness (>70%), chemotaxonomic, and molecular profile, P. pseudoalcaligenes ATCC 17440(T) and P. oleovorans ATCC 8062(T) should be united under the same name; according to the rules of priority, P. oleovorans, the first described species, is the earlier synonym and P. pseudoalcaligenes is the later synonym. As a consequence, the division of the species P. oleovorans into two novel subspecies is proposed: P. oleovorans subsp. oleovorans subsp. nov. (type strain ATCC 8062(T) = DSM 1045(T) = NCIB 6576(T)), P. oleovorans subsp. lubricantis subsp. nov. (type strain RS1(T) = ATCC BAA-1494(T) = DSM 21016(T)). PMID:19936829

  1. ATCC 43642 replaces ATCC 23581 as the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926. Opinion 91. Judicial Commission of the International Committee on Systematics of Prokaryotes.

    PubMed

    Tindall, B J

    2014-10-01

    The Judicial Commission affirms that, according to information presented to it, the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926 designated on the Approved Lists of Bacterial Names (ATCC 23581) has been shown not to represent an authentic culture of strain RGA (a member of the serovar Icterohaemorrhagiae) and ATCC 43642, derived from an authentic strain of strain RGA, a member of the serovar Icterohaemorrhagiae, is designated the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926.

  2. Growth of Lactobacillus paracasei ATCC334 in a cheese model system: A biochemical approach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Growth of Lactobacillus paracasei ATCC 334, in a cheese-ripening model system based upon a medium prepared from ripening Cheddar cheese extract (CCE) was evaluated. Lactobacillus paracasei ATCC 334 grows in CCE made from cheese ripened for 2 (2mCCE), 6 (6mCCE), and 8 (8mCCE) mo, to final cell densit...

  3. Complete Genome Sequence of Arthrobacter sp. ATCC 21022, a Host for Bacteriophage Discovery

    PubMed Central

    Russell, Daniel A.

    2016-01-01

    We report the complete genome sequence of Arthrobacter sp. ATCC 21022, a strain maintained by ATCC and a commonly used host for bacteriophage isolation and genomic analysis. The strain is prophage-free and CRISPR-free but codes for two predicted restriction-modification systems. PMID:27013048

  4. Identification of the Herboxidiene Biosynthetic Gene Cluster in Streptomyces chromofuscus ATCC 49982

    PubMed Central

    Shao, Lei; Zi, Jiachen; Zeng, Jia

    2012-01-01

    The 53-kb biosynthetic gene cluster for the novel anticholesterol natural product herboxidiene was identified in Streptomyces chromofuscus ATCC 49982 by genome sequencing and gene inactivation. In addition to herboxidiene, a biosynthetic intermediate, 18-deoxy-herboxidiene, was also isolated from the fermentation broth of S. chromofuscus ATCC 49982 as a minor metabolite. PMID:22247174

  5. Complete Genome Sequence of the Type Strain of Aeromonas schubertii, ATCC 43700

    PubMed Central

    Liu, Lihui; Zhang, Defeng; Fu, Xiaozhe; Shi, Cunbin; Lin, Qiang

    2016-01-01

    We sequenced the complete genome of the type strain of Aeromonas schubertii, ATCC 43700. The full genome sequence of A. schubertii ATCC 43700 is 4,356,858 bp, which encodes 3,842 proteins and contains 110 predicted RNA genes. PMID:26893413

  6. Tissue reaction and immunity in swine immunized with Actinobacillus pleuropneumoniae vaccines.

    PubMed

    Willson, P J; Rossi-Campos, A; Potter, A A

    1995-10-01

    These studies were done to develop a subunit vaccine for swine that would protect against disease, but not create unacceptable tissue reactions at the immunization site. Swine were used to evaluate the local effects of subunit vaccines prepared from extracts of Actinobacillus pleuropneumoniae serotype 1 containing one of a wide variety of adjuvants. The antigen was an anionic fraction of a saline extract of A. pleuropneumoniae (ANEX). The adjuvants used were vegetable oils (peanut, sesame, canola, or corn oils, vitamin E, or Lipposyn II emulsion); mineral oil (Marcol-52) and other materials (aluminum hydroxide, polyethylene glycol, Quil-A, Amphigen, or Emulsigen-Plus). Two types of experiments were done. In the 1st set of experiments, pigs were given multiple simultaneous injections in different sites and euthanized on days 1, 3, 7, 14, 21, or 28. Tissues were examined for gross and histopathological lesions. In the 2nd set of experiments, 48 pigs were allocated to 6 groups and vaccinated twice with a vaccine containing ANEX antigen combined with one of various adjuvants. Antibody responses and protection from challenge were evaluated. Among the adjuvants that were tested, mineral oils induced protective immunity, although the mineral oil Marcol-52 resulted in severe tissue reactions. The vegetable oils induced little protective immunity, and some of them were quite irritating. The response to the other materials ranged from little irritation or protection induced by the vaccine containing aluminum hydroxide to effective protection without irritation after vaccination with ANEX/Amphigen or ANEX/Emulsigen-Plus combinations. In conclusion, swine were protected against disease by a subunit vaccine that did not create unacceptable tissue reaction at the immunization site.

  7. Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catechol siderophores.

    PubMed Central

    Diarra, M S; Dolence, J A; Dolence, E K; Darwish, I; Miller, M J; Malouin, F; Jacques, M

    1996-01-01

    Siderophores bind ferric ions and are involved in receptor-specific iron transport into bacteria. Six types of siderophores were tested against strains representing the 12 different serotypes of Actinobacillus pleuropneumoniae. Ferrichrome and bis-catechol-based siderophores showed strong growth-promoting activities for A. pleuropneumoniae in a disk diffusion assay. Most strains of A. pleuropneumoniae tested were able to use ferrichrome (21 of 22 or 95%), ferrichrome A (20 of 22 or 90%), and lysine-based bis-catechol (20 of 22 or 90%), while growth of 36% (8 of 22) was promoted by a synthetic hydroxamate, N5-acetyl-N5-hydroxy-L-ornithine tripeptide. A. pleuropneumoniae serotype 1 (strain FMV 87-682) and serotype 5 (strain 2245) exhibited a distinct yellow halo around colonies on Chrome Azurol S agar plates, suggesting that both strains can produce an iron chelator (siderophore) in response to iron stress. The siderophore was found to be neither a phenolate nor a hydroxamate by the chemical tests of Arnow and Csaky, respectively. This is the first report demonstrating the production of an iron chelator and the use of exogenous siderophores by A. pleuropneumoniae. A spermidine-based bis-catechol siderophore conjugated to a carbacephalosporin was shown to inhibit growth of A. pleuropneumoniae. A siderophore-antibiotic-resistant strain was isolated and shown to have lost the ability to use ferrichrome, synthetic hydroxamate, or catechol-based siderophores when grown under conditions of iron restriction. This observation indicated that a common iron uptake pathway, or a common intermediate, for hydroxamate- and catechol-based siderophores may exist in A. pleuropneumoniae. PMID:8975614

  8. A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

    PubMed Central

    Rossi, Ciro C.; Bossé, Janine T.; Li, Yanwen; Witney, Adam A.; Gould, Kate A.; Langford, Paul R.; Bazzolli, Denise M.S.

    2016-01-01

    Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies. PMID:27402897

  9. Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8.

    PubMed

    Li, Gang; Xie, Fang; Zhang, Yanhe; Bossé, Janine T; Langford, Paul R; Wang, Chunlai

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae. PMID:26509499

  10. Sub-inhibitory concentrations of penicillin G induce biofilm formation by field isolates of Actinobacillus pleuropneumoniae.

    PubMed

    Hathroubi, S; Fontaine-Gosselin, S-È; Tremblay, Y D N; Labrie, J; Jacques, M

    2015-09-30

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium and causative agent of porcine pleuropneumonia. This is a highly contagious disease that causes important economic losses to the swine industry worldwide. Penicillins are extensively used in swine production and these antibiotics are associated with high systemic clearance and low oral bioavailability. This may expose A. pleuropneumoniae to sub-inhibitory concentrations of penicillin G when the antibiotic is administered orally. Our goal was to evaluate the effect of sub-minimum inhibitory concentration (MIC) of penicillin G on the biofilm formation of A. pleuropneumoniae. Biofilm production of 13 field isolates from serotypes 1, 5a, 7 and 15 was tested in the presence of sub-MIC of penicillin G using a polystyrene microtiter plate assay. Using microscopy techniques and enzymatic digestion, biofilm architecture and composition were also characterized after exposure to sub-MIC of penicillin G. Sub-MIC of penicillin G significantly induced biofilm formation of nine isolates. The penicillin G-induced biofilms contained more poly-N-acetyl-D-glucosamine (PGA), extracellular DNA and proteins when compared to control biofilms grown without penicillin G. Additionally, penicillin G-induced biofilms were sensitive to DNase which was not observed with the untreated controls. Furthermore, sub-MIC of penicillin G up-regulated the expression of pgaA, which encodes a protein involved in PGA synthesis, and the genes encoding the envelope-stress sensing two-component regulatory system CpxRA. In conclusion, sub-MICs of penicillin G significantly induce biofilm formation and this is likely the result of a cell envelope stress sensed by the CpxRA system resulting in an increased production of PGA and other matrix components.

  11. Role of high-avidity binding of human neutrophil myeloperoxidase in the killing of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Miyasaki, K T; Zambon, J J; Jones, C A; Wilson, M E

    1987-01-01

    The binding of the neutrophil enzyme myeloperoxidase (MPO) to microbial surfaces is believed to be the first step in its microbicidal activity. The MPO-H2O2-Cl- system is responsible for most oxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. There appear to be three forms of MPO (MPO I, II, and III), all of which can kill this organism in the presence of H2O2 and chloride. In this study, we characterized the binding of native human neutrophil MPO to A. actinomycetemcomitans by an elution procedure dependent on the cationic detergent cetyltrimethylammonium bromide. Binding of native MPO was rapid and reached apparent equilibrium within 1 min. A proportion of binding under equilibrium conditions was saturable and highly avid, with a capacity of 4,500 sites per cell and a dissociation constant of 7.9 X 10(-10) M. At equal protein concentrations, more MPO III bound than MPO II, and more MPO II bound than MPO I. The high-avidity interaction was inhibitable with yeast mannan and with the serotype-defining mannan of A. actinomycetemcomitans. Binding was also partially reversible with yeast mannan. MPO bound to the high-avidity sites did not oxidize guaiacol but oxidized chloride, as detected by the chlorination of taurine. MPO bound to the high-avidity sites was incapable of killing A. actinomycetemcomitans alone in the presence of H2O2 and Cl-, but potentiated killing when sufficient additional MPO was provided. The killing of A. actinomycetemcomitans by the MPO-H2O2-Cl- system was inhibited by yeast mannan and a serotype-defining mannan of A. actinomycetemcomitans. We conclude that high-avidity binding of MPO to the surface of A. actinomycetemcomitans is a mannan-specific interaction and that MPO bound to the high-avidity sites is essential but not alone sufficient to kill A. actinomycetemcomitans. PMID:3032796

  12. In vitro activity of antibiotics alone and in combination against Actinobacillus actinomycetemcomitans.

    PubMed

    Yogev, R; Shulman, D; Shulman, S T; Glogowski, W G

    1986-01-01

    The MICs for 90% of the organisms tested (MIC90S) of 11 antibiotics against 24 clinical isolates of Actinobacillus actinomycetemcomitans were determined by the MIC 2000 system. The lowest MIC90S (16 micrograms/ml) were observed with ceftriaxone and rifampin. The next lowest MIC90S were found with cephapirin, tetracycline, and chloramphenicol (3.12 micrograms/ml). The MIC90S of penicillin, ampicillin, ticarcillin, piperacillin, and amikacin were each greater than or equal to 12.5 micrograms/ml. Antibiotic synergy was studied by the killing curve method and was defined as a greater than or equal to 2 log10 reduction in CFU when two antibiotics were used in combination at one-fourth the MBC for each compared with the effect of each antibiotic alone at one-half the MBC. Synergism between rifampin and penicillin, cephapirin, or ceftriaxone was tested for with 12 A. actinomycetemcomitans strains. In 7 of 37 instances, synergism was demonstrated for the combinations rifampin plus ceftriaxone (n = 3) or rifampin plus penicillin (n = 4); in 9 instances, an additive effect was noted, and impaired killing with drug combinations compared with the effect of a single antibiotic was suggested in 4 strains. The majority of strains were indifferent to the combinations. Similarly, variable results were observed when the combination of trimethoprim and cephapirin was tested against eight A. actinomycetemcomitans strains. Our data suggest that rifampin and cephapirin are the most active of the 11 antibiotics studied against A. actinomycetemcomitans. In addition, in vitro synergism between rifampin and other antibiotics or between trimethoprim and cephapirin was not consistently demonstrable.

  13. Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8.

    PubMed

    Li, Gang; Xie, Fang; Zhang, Yanhe; Bossé, Janine T; Langford, Paul R; Wang, Chunlai

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae.

  14. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

    PubMed Central

    Hathroubi, S.; Hancock, M. A.; Langford, P. R.; Tremblay, Y. D. N.; Labrie, J.

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

  15. Experimental model of swine pneumonic pasteurellosis using crude Actinobacillus pleuropneumoniae cytotoxin and Pasteurella multocida given endobronchially.

    PubMed Central

    Chung, W B; Bäckström, L R; Collins, M T

    1994-01-01

    This study was designed to develop and characterize a swine pneumonic pasteurellosis model by concurrent introduction of Pasteurella multocida type A and Actinobacillus pleuropneumoniae crude cytotoxin. After a series of preliminary experiments, a combination of 4 x 10(9) P. multocida and 4,000 toxic units of A. pleuropneumoniae crude cytotoxin was determined to produce optimal results. A total of 48 pigs were divided into four groups of 12 pigs each. The control group received buffered saline only. Four pigs from each group were randomly selected for necropsy 3, 7 and 14 days postinoculation (PI). Inoculation of pigs with P. multocida and A. pleuropneumoniae cytotoxin (group 1) resulted in moderate to severe pneumonia. Pasteurella multocida was isolated from pneumonic lesions, grossly normal lung, and bronchial lymph nodes of all group 1 pigs throughout the 14 day experimental period. Pathological changes typical of field cases of swine pneumonic pasteurellosis were produced. Pigs inoculated with P. multocida alone (group 2) had pneumonic lesions and P. multocida was reisolated from lungs at three days PI. Pasteurella multocida was not isolated from these pigs at 7 and 14 days PI, except for one pig in which an abscess developed in the thorax. Pulmonary lesions induced by A. pleuropneumoniae crude cytotoxin alone (group 3) were transient and resolved by seven days PI. Group 1 pigs had significantly greater lung lesion volumes than group 2 and 3 pigs at 3, 7 and 14 days PI. Statistical analysis indicated a significant interactive effect of P. multocida and A. pleuropneumoniae cytotoxin on the development of lung lesion volumes at 7 and 14 days PI (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. PMID:8143249

  16. Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8

    PubMed Central

    Li, Gang; Xie, Fang; Zhang, Yanhe; Bossé, Janine T.; Langford, Paul R.; Wang, Chunlai

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5’-diphosphate 3’-diphosphate (ppGpp) and guanosine 5’-triphosphate 3’-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae. PMID:26509499

  17. Tissue reaction and immunity in swine immunized with Actinobacillus pleuropneumoniae vaccines.

    PubMed Central

    Willson, P J; Rossi-Campos, A; Potter, A A

    1995-01-01

    These studies were done to develop a subunit vaccine for swine that would protect against disease, but not create unacceptable tissue reactions at the immunization site. Swine were used to evaluate the local effects of subunit vaccines prepared from extracts of Actinobacillus pleuropneumoniae serotype 1 containing one of a wide variety of adjuvants. The antigen was an anionic fraction of a saline extract of A. pleuropneumoniae (ANEX). The adjuvants used were vegetable oils (peanut, sesame, canola, or corn oils, vitamin E, or Lipposyn II emulsion); mineral oil (Marcol-52) and other materials (aluminum hydroxide, polyethylene glycol, Quil-A, Amphigen, or Emulsigen-Plus). Two types of experiments were done. In the 1st set of experiments, pigs were given multiple simultaneous injections in different sites and euthanized on days 1, 3, 7, 14, 21, or 28. Tissues were examined for gross and histopathological lesions. In the 2nd set of experiments, 48 pigs were allocated to 6 groups and vaccinated twice with a vaccine containing ANEX antigen combined with one of various adjuvants. Antibody responses and protection from challenge were evaluated. Among the adjuvants that were tested, mineral oils induced protective immunity, although the mineral oil Marcol-52 resulted in severe tissue reactions. The vegetable oils induced little protective immunity, and some of them were quite irritating. The response to the other materials ranged from little irritation or protection induced by the vaccine containing aluminum hydroxide to effective protection without irritation after vaccination with ANEX/Amphigen or ANEX/Emulsigen-Plus combinations. In conclusion, swine were protected against disease by a subunit vaccine that did not create unacceptable tissue reaction at the immunization site. PMID:8548692

  18. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae.

    PubMed

    Hathroubi, S; Hancock, M A; Bossé, J T; Langford, P R; Tremblay, Y D N; Labrie, J; Jacques, M

    2016-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

  19. Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

    PubMed Central

    Labrie, Josée; Hernandez Reyes, Yenney; Burciaga Nava, Jorge A.; Gagnon, Carl A.; Jacques, Mario

    2014-01-01

    Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools. PMID:24878741

  20. Absence of TolC Impairs Biofilm Formation in Actinobacillus pleuropneumoniae by Reducing Initial Attachment

    PubMed Central

    Yuan, Jianlin; Lau, Gee W.; Wen, Yiping; Wu, Rui; Zhao, Qin; Huang, Xiaobo; Yan, Qigui; Huang, Yong; Wen, Xintian

    2016-01-01

    Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a major cause of economic loss in swine industry worldwide. TolC, the key component of multidrug efflux pumps and type I secretion systems, has been well-studied as an exit duct for numerous substances in many Gram-negative bacteria. By contrast, little is known on the role of TolC in biofilm formation. In this study, a ΔtolC mutant was used to examine the importance of TolC in biofilm formation of A. pleuropneumoniae. Surface attachment assays demonstrated the essential role of TolC in initial attachment of biofilm cells. The loss of TolC function altered surface hydrophobicity, and resulted in greatly reduced autoaggregation in ΔtolC. Using both enzymatic treatments and confocal microscopy, biofilm composition and architecture were characterized. When compared against the wild-type strain, the poly-β-1, 6-N-acetyl-D-glucosamine (PGA), an important biofilm matrix component of A. pleuropneumoniae, was significantly reduced at the initial attachment stage in ΔtolC. These results were confirmed by mRNA level using quantitative RT-PCR. Additionally, defective secretion systems in ΔtolC may also contribute to the deficiency in biofilm formation. Taken together, the current study demonstrated the importance of TolC in the initial biofilm formation stage in A. pleuropneumoniae. These findings could have important clinical implications in developing new treatments against biofilm-related infections by A. pleuropneumoniae. PMID:27681876

  1. Nonspecific Adherence by Actinobacillus actinomycetemcomitans Requires Genes Widespread in Bacteria and Archaea

    PubMed Central

    Kachlany, Scott C.; Planet, Paul J.; Bhattacharjee, Mrinal K.; Kollia, Evyenia; DeSalle, Rob; Fine, Daniel H.; Figurski, David H.

    2000-01-01

    The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903φkan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad− mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad

  2. Isolation and genetic characterization of an Actinobacillus pleuropneumoniae serovar K12:O3 strain.

    PubMed

    Ito, Hiroya; Matsumoto, Atsuko

    2015-01-01

    An atypical Actinobacillus pleuropneumoniae serovar 12 strain, termed QAS106, was isolated from a clinical case of porcine pleuropneumonia in Japan. An immunodiffusion (ID) test identified the strain as serovar 12. However, the ID test also demonstrated that strain QAS106 shared antigenic determinants with both the serovar 3 and 15 reference strains. Strain QAS106 was positive in the capsular serovar 12-specific polymerase chain reaction (PCR) assay, while the PCR toxin gene profiling and omlA PCR typing assays indicated that strain QAS106 was similar to serovar 3. The nucleotide sequence of the 16S ribosomal DNA (rDNA) of strain QAS106 was identical with that of serovars 3 and 12, but it showed 99.7% identity with that of serovar 15. Nucleotide sequence analysis revealed that genes involved in biosynthesis of the capsular polysaccharide (CPS) of strain QAS106 were identical to those of serovar 12 at the amino acid level. On the other hand, strain QAS106 would express putative proteins involved in the biosynthesis of lipopolysaccharide (LPS) O-polysaccharide (O-PS), the amino acid sequences of which were identical or nearly identical to those of serovars 3 and 15. In conclusion, strain QAS106 should be recognized as K12:O3, even though typical serovar 12 strains are K12:O12. The emergence of an atypical A. pleuropneumoniae serovar 12 strain expressing a rare combination of CPS and O-PS antigens would hamper precise serodiagnosis by the use of either CPS- or LPS-based serodiagnostic methodology alone. PMID:25387844

  3. Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073▿

    PubMed Central

    Li, Qingyan; Seffernick, Jennifer L.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2009-01-01

    Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications. PMID:19767460

  4. Complete genome sequence of Anabaena variabilis ATCC 29413

    SciTech Connect

    Thiel, Teresa; Pratte, Brenda S.; Zhong, Jinshun; Goodwin, Lynne A.; Copeland, A; Lucas, Susan; Han, Cliff; Pitluck, Sam; Land, Miriam L; Kyrpides, Nikos C; Woyke, Tanja

    2013-01-01

    Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Ana-baena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40 C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.

  5. Complete genome sequence of Anabaena variabilis ATCC 29413.

    PubMed

    Thiel, Teresa; Pratte, Brenda S; Zhong, Jinshun; Goodwin, Lynne; Copeland, Alex; Lucas, Susan; Han, Cliff; Pitluck, Sam; Land, Miriam L; Kyrpides, Nikos C; Woyke, Tanja

    2014-06-15

    Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40(°) C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence. PMID:25197444

  6. Biotransformation of (-)beta-pinene by Aspergillus niger ATCC 9642.

    PubMed

    Toniazzo, Geciane; de Oliveira, Débora; Dariva, Cláudio; Oestreicher, Enrique Guillermo; Antunes, Octávio A C

    2005-01-01

    The main objective of this work was to investigate the biotransformations of (-)alpha-pinene, (-)beta-pinene, and (+) limonene by Aspergillus niger ATCC 9642. The culture conditions involved--concentration of cosolvent (EtOH), substrate applied, and sequential addition of substrates were--investigated. Adaptation of the precultures with small amounts of substrate was also studied. The experiments were performed in conical flasks with liquid cultures. This strain of A. niger was able to convert only (-)beta-pinene into alpha-terpineol. An optimum conversion of (-)beta-pinene into alpha-terpineol of about 4% was obtained when the substrate was applied as a diluted solution in EtOH and sequential addition of substrate was used.

  7. Experimental Identification of Actinobacillus pleuropneumoniae Strains L20 and JL03 Heptosyltransferases, Evidence for a New Heptosyltransferase Signature Sequence

    PubMed Central

    Merino, Susana; Knirel, Yuriy A.; Regué, Miguel; Tomás, Juan M.

    2013-01-01

    We experimentally identified the activities of six predicted heptosyltransferases in Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose (D,D-Hep) residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D261 for S261, being unique. PMID:23383222

  8. Genome Sequence of the Ethanol-Producing Zymomonas mobilis subsp. mobilis Lectotype Strain ATCC 10988 ▿

    PubMed Central

    Pappas, Katherine M.; Kouvelis, Vassili N.; Saunders, Elizabeth; Brettin, Thomas S.; Bruce, David; Detter, Chris; Balakireva, Mariya; Han, Cliff S.; Savvakis, Giannis; Kyrpides, Nikos C.; Typas, Milton A.

    2011-01-01

    Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome. PMID:21725006

  9. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin.

    PubMed

    Sager, Martin; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Benga, Laurentiu

    2015-01-01

    [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms

  10. A Transcriptome Map of Actinobacillus pleuropneumoniae at Single-Nucleotide Resolution Using Deep RNA-Seq.

    PubMed

    Su, Zhipeng; Zhu, Jiawen; Xu, Zhuofei; Xiao, Ran; Zhou, Rui; Li, Lu; Chen, Huanchun

    2016-01-01

    Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumoniae, a highly contagious respiratory disease of swine. Although the genome of A. pleuropneumoniae was sequenced several years ago, limited information is available on the genome-wide transcriptional analysis to accurately annotate the gene structures and regulatory elements. High-throughput RNA sequencing (RNA-seq) has been applied to study the transcriptional landscape of bacteria, which can efficiently and accurately identify gene expression regions and unknown transcriptional units, especially small non-coding RNAs (sRNAs), UTRs and regulatory regions. The aim of this study is to comprehensively analyze the transcriptome of A. pleuropneumoniae by RNA-seq in order to improve the existing genome annotation and promote our understanding of A. pleuropneumoniae gene structures and RNA-based regulation. In this study, we utilized RNA-seq to construct a single nucleotide resolution transcriptome map of A. pleuropneumoniae. More than 3.8 million high-quality reads (average length ~90 bp) from a cDNA library were generated and aligned to the reference genome. We identified 32 open reading frames encoding novel proteins that were mis-annotated in the previous genome annotations. The start sites for 35 genes based on the current genome annotation were corrected. Furthermore, 51 sRNAs in the A. pleuropneumoniae genome were discovered, of which 40 sRNAs were never reported in previous studies. The transcriptome map also enabled visualization of 5'- and 3'-UTR regions, in which contained 11 sRNAs. In addition, 351 operons covering 1230 genes throughout the whole genome were identified. The RNA-Seq based transcriptome map validated annotated genes and corrected annotations of open reading frames in the genome, and led to the identification of many functional elements (e.g. regions encoding novel proteins, non-coding sRNAs and operon structures). The transcriptional units described in this study

  11. The survival rate of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides forsythus following 4 randomized treatment modalities.

    PubMed

    Shiloah, J; Patters, M R; Dean, J W; Bland, P; Toledo, G

    1997-08-01

    The overall goal of this clinical study was to determine the short-term anti-infective effects of four randomized treatment modalities on Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Bacteroides forsythus (Bf) and determine the effects of bacterial survival on treatment outcomes in patients with adult periodontitis. Twelve adult patients requiring therapy for moderate periodontitis were selected for this study. All patients had at least one tooth in each quadrant that had an inflamed pocket of probing depth > or =5 mm with probing attachment loss that harbored at least one of the following three periodontal pathogens: Aa, Pg, or Bf. The number of target organisms per site was determined pre-operatively, at 1 week, and 1 month and 3 months postoperatively utilizing DNA probes. One quadrant in each patient was randomly assigned to each one of the following four treatment groups: 1) scaling and root planing (SRP group); 2) pocket reduction through osseous surgery and apically-positioned flap (OS group); 3) modified Widman flap (MWF group); and 4) modified Widman flap and topical application of saturated citric acid at pH 1 for 3 minutes (CA group). The 4 treatment modalities were performed in one appointment. No postoperative antibiotics were used. Patients were instructed to supplement their daily oral hygiene with chlorohexidine oral rinse during the study. The results of this investigation indicated that: 1) none of the treatment modalities was effective in eliminating the target species; 2) the incidence of infected sites for all groups was 100% preoperatively; 62.5%, 33.3%, and 31.3% at 1 week, and 1 and 3 months postoperatively, respectively; 3) these infected sites lost 1.1 +/- 0.4 mm of probing attachment compared to gain of 0.0 +/- 0.3 mm for uninfected sites; 4) the infected sites had higher plaque and bleeding on probing 0.9 +/- 0.3, 73 +/- 12%, respectively, compared to 0.3 +/- 0.1 and 30 +/- 8% for the uninfected sites

  12. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin

    PubMed Central

    Sager, Martin; Benten, W. Peter M.; Engelhardt, Eva; Gougoula, Christina; Benga, Laurentiu

    2015-01-01

    [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms

  13. A Transcriptome Map of Actinobacillus pleuropneumoniae at Single-Nucleotide Resolution Using Deep RNA-Seq

    PubMed Central

    Su, Zhipeng; Zhu, Jiawen; Xu, Zhuofei; Xiao, Ran; Zhou, Rui; Li, Lu; Chen, Huanchun

    2016-01-01

    Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumoniae, a highly contagious respiratory disease of swine. Although the genome of A. pleuropneumoniae was sequenced several years ago, limited information is available on the genome-wide transcriptional analysis to accurately annotate the gene structures and regulatory elements. High-throughput RNA sequencing (RNA-seq) has been applied to study the transcriptional landscape of bacteria, which can efficiently and accurately identify gene expression regions and unknown transcriptional units, especially small non-coding RNAs (sRNAs), UTRs and regulatory regions. The aim of this study is to comprehensively analyze the transcriptome of A. pleuropneumoniae by RNA-seq in order to improve the existing genome annotation and promote our understanding of A. pleuropneumoniae gene structures and RNA-based regulation. In this study, we utilized RNA-seq to construct a single nucleotide resolution transcriptome map of A. pleuropneumoniae. More than 3.8 million high-quality reads (average length ~90 bp) from a cDNA library were generated and aligned to the reference genome. We identified 32 open reading frames encoding novel proteins that were mis-annotated in the previous genome annotations. The start sites for 35 genes based on the current genome annotation were corrected. Furthermore, 51 sRNAs in the A. pleuropneumoniae genome were discovered, of which 40 sRNAs were never reported in previous studies. The transcriptome map also enabled visualization of 5'- and 3'-UTR regions, in which contained 11 sRNAs. In addition, 351 operons covering 1230 genes throughout the whole genome were identified. The RNA-Seq based transcriptome map validated annotated genes and corrected annotations of open reading frames in the genome, and led to the identification of many functional elements (e.g. regions encoding novel proteins, non-coding sRNAs and operon structures). The transcriptional units described in this study

  14. Risk assessment of transmission of capsule-deficient, recombinant Actinobacillus pleuropneumoniae.

    PubMed

    Inzana, Thomas J; Glindemann, Gretchen; Fenwick, Bradley; Longstreth, Janice; Ward, Daniel

    2004-11-30

    Actinobacillus pleuropneumoniae is the etiologic agent of swine pleuropneumonia. Live, non-encapsulated vaccine strains have been shown to be efficacious in preventing acute disease in pigs. Recombinant DNA technology has the advantage of generating defined mutants that are safe, but maintain critical immunoprotective components. However, some recombinant strains have the disadvantage of containing antibiotic resistance genes that could be transferred to the animal's normal bacterial flora. Using DNA allelic exchange we have constructed attenuated, capsule-deficient mutants of A. pleuropneumoniae that contain a kanamycin resistance (Kn(R)) gene within the capsule locus of the genome. Following intranasal or intratracheal challenge of pigs the encapsulated parent strains colonized the challenge pigs, and were transmitted to contact pigs. In contrast, the capsule-deficient mutants were recovered only from the challenged pigs and not from contact pigs. Each kanamycin-resistant colony type recovered from the respiratory or gastrointestinal tracts of pigs challenged with the recombinant strain was screened with a probe specific for the Kn(R) gene. All probe-positive colonies were assayed for the specific Kn(R) gene by amplification of a 0.9 kb fragment of the antibiotic resistance gene by PCR. The 0.9 kb fragment was amplified from the recombinant A. pleuropneumoniae colonies, but not from any of the heterologous bacteria, indicating there was no evidence of transmission of the Kn(R) gene to resident bacteria. Following aerosol exposure of 276 pigs with recombinant, non-encapsulated A. pleuropneumoniae the recombinant bacteria were not recovered from any nasal swabs of 75 pigs tested or environmental samples 18 h after challenge. Statistical risk analysis, based on the number of kanamycin-resistant colonies screened, indicated that undetected transmission of the Kn(R) gene could still have occurred in at most 1.36% of kanamycin-resistant bacteria in contact with

  15. Evaluation and Field Validation of PCR Tests for Detection of Actinobacillus pleuropneumoniae in Subclinically Infected Pigs

    PubMed Central

    Fittipaldi, Nahuel; Broes, André; Harel, Josée; Kobisch, Marylène; Gottschalk, Marcelo

    2003-01-01

    Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (109 to 102 CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (102 CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at −20°C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (κ = 0.65) was found between the presence of A

  16. Proteomic and immunoproteomic characterization of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae

    PubMed Central

    2011-01-01

    Background Protection of pigs by vaccination against Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is hampered by the presence of 15 different serotypes. A DIVA subunit vaccine comprised of detergent-released proteins from A. pleuropneumoniae serotypes 1, 2 and 5 has been developed and shown to protect pigs from clinical symptoms upon homologous and heterologous challenge. This vaccine has not been characterized in-depth so far. Thus we performed i) mass spectrometry in order to identify the exact protein content of the vaccine and ii) cross-serotype 2-D immunoblotting in order to discover cross-reactive antigens. By these approaches we expected to gain results enabling us to argue about the reasons for the efficacy of the analyzed vaccine. Results We identified 75 different proteins in the vaccine. Using the PSORTb algorithm these proteins were classified according to their cellular localization. Highly enriched proteins are outer membrane-associated lipoproteins like OmlA and TbpB, integral outer membrane proteins like FrpB, TbpA, OmpA1, OmpA2, HgbA and OmpP2, and secreted Apx toxins. The subunit vaccine also contained large amounts of the ApxIVA toxin so far thought to be expressed only during infection. Applying two-dimensional difference gel electrophoresis (2-D DIGE) we showed different isoforms and variations in expression levels of several proteins among the strains used for vaccine production. For detection of cross-reactive antigens we used detergent released proteins of serotype 7. Sera of pigs vaccinated with the detergent-released proteins of serotypes 1, 2, and 5 detected seven different proteins of serotype 7, and convalescent sera of pigs surviving experimental infection with serotype 7 reacted with 13 different proteins of the detergent-released proteins of A. pleuropneumoniae serotypes 1, 2, and 5. Conclusions A detergent extraction-based subunit vaccine of A. pleuropneumoniae was characterized by mass

  17. Site-specific subgingival colonization by Actinobacillus actinomycetemcomitans in orthodontic patients.

    PubMed

    Paolantonio, M; Festa, F; di Placido, G; D'Attilio, M; Catamo, G; Piccolomini, R

    1999-04-01

    A high prevalence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque in patients for orthodontia already has been observed. The present study had the following aims: 1) to ascertain a direct relationship between the orthodontic appliance placement and the subgingival colonization by Aa, and 2) to determine whether the Aa growth specifically occurred on teeth with braces attached or whether the presence of orthodontic appliances could also cause the isolation of Aa in teeth free from therapeutic appliances. Twenty-four young systemically and periodontally healthy subjects with malaligned and crowded teeth in the anterior sextants of both dental arches participated in this study. After 1 session of ultrasonic scaling with oral hygiene instructions during the first experimental session, the mesiobuccal sites of the first molars and the distobuccal sites of the lateral incisors in both dental arches in each participant were subjected to clinical and microbiologic examination for the recovery of Aa. Clinical examination consisted of recording the presence of plaque and the examination of gingival bleeding on probing and probing depth. Microbiologic sampling was obtained with the insertion of 3 sterile paper points at the deepest part of each gingival sulcus. Altogether, 192 periodontal sites were examined. After the examinations, the patients received fixed orthodontic appliances in only 1 dental arch (test sites) and the other one was left free from appliances (control sites). Clinical examination and microbiologic sampling were repeated in the same experimental test and control sites after 4, 8, and 12 weeks. At the 12-week session, the orthodontic appliance was removed from the test arch, and, 4 weeks later, a further clinical and microbiologic examination was performed. The results showed that, during the period with orthodontic appliances, the presence of plaque scores and the gingival bleeding on probing scores were increased significantly and that

  18. Dielectric characterization of forespores isolated from Bacillus megaterium ATCC 19213.

    PubMed Central

    Marquis, R E; Bender, G R; Carstensen, E L; Child, S Z

    1983-01-01

    Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous suspensions were nearly as dehydrated as mature spores, as indicated by low dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The forespores lacked dipicolinate, had only minimal levels of calcium, magnesium, manganese, potassium, and sodium, and were more heat sensitive than vegetative cells. The effective homogeneous conductivities and dielectric constants measured over a frequency range of 1 to 200 MHz indicated that the inherent conductivities of the forespores were unusually low, in keeping with their low mineral contents, but that the forespores could be invaded by environmental ions which could penetrate dielectrically effective membranes. Overall, our findings support the view that the dehydration of a forespore during stage III of sporogenesis may be the result of ion movements out of the forespore into the sporangium. PMID:6401285

  19. Microcalorimetric study of cellulose degradation by Cellulomonas uda ATCC 21399

    SciTech Connect

    Dermoun, Z.; Belaich, J.P.

    1985-07-01

    A newly designed batch calorimeter was used to investigate the degradability of some celluloses having varying degrees of crystallinity. The PTC of an aerobic culture of Cellulomonas uda ATCC 21399 obtained revealed a diauxic growth which is attributed to the presence of hemicellulose contaminating Avicel and MN300 cellulose. The microcrystalline celluloses used were not completely utilized, whereas amorphous cellulose was easily metabolized, indicating that under the growth conditions used here, the physical structure of cellulose strongly influenced its microbial degradability. An equivalent growth yield of ca. 0.44 g/g was found with all the substrates used. The heat evolved by metabolism of one g cellulose was - 5.86 kJ/g, a value similar to that obtained with glucose culture. The growth rate was the only variable parameter. The data obtained showed as expected that the hydrolysis product of cellulose was consumed in the same way as that of glucose and that the only limiting factor to the biodegradability of cellulose was the breakdown of the polymeric substrate. It is concluded that data obtained with glucose metabolism can be used to evaluate the extent of cellulose degradation.

  20. L-Lactic Acid Production by Lactobacillus rhamnosus ATCC 10863

    PubMed Central

    Senedese, Ana Lívia Chemeli; Maciel Filho, Rubens; Maciel, Maria Regina Wolf

    2015-01-01

    Lactic acid has been shown to have the most promising application in biomaterials as poly(lactic acid). L. rhamnosus ATCC 10863 that produces L-lactic acid was used to perform the fermentation and molasses was used as substrate. A solution containing 27.6 g/L of sucrose (main composition of molasses) and 3.0 g/L of yeast extract was prepared, considering the final volume of 3,571 mL (14.0% (v/v) inoculum). Batch and fed batch fermentations were performed with temperature of 43.4°C and pH of 5.0. At the fed batch, three molasses feed were applied at 12, 24, and 36 hours. Samples were taken every two hours and the amounts of lactic acid, sucrose, glucose, and fructose were determined by HPLC. The sucrose was barely consumed at both processes; otherwise the glucose and fructose were almost entirely consumed. 16.5 g/L of lactic acid was produced at batch and 22.0 g/L at fed batch. Considering that lactic acid was produced due to the low concentration of the well consumed sugars, the final amount was considerable. The cell growth was checked and no substrate inhibition was observed. A sucrose molasses hydrolysis is suggested to better avail the molasses fermentation with this strain, surely increasing the L-lactic acid. PMID:25922852

  1. New insights into chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712.

    PubMed

    Fernández-Martínez, Lorena T; Borsetto, Chiara; Gomez-Escribano, Juan Pablo; Bibb, Maureen J; Al-Bassam, Mahmoud M; Chandra, Govind; Bibb, Mervyn J

    2014-12-01

    Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916-sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.

  2. New Insights into Chloramphenicol Biosynthesis in Streptomyces venezuelae ATCC 10712

    PubMed Central

    Fernández-Martínez, Lorena T.; Borsetto, Chiara; Gomez-Escribano, Juan Pablo; Bibb, Maureen J.; Al-Bassam, Mahmoud M.; Chandra, Govind

    2014-01-01

    Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916–sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na+/H+ antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway. PMID:25267678

  3. Conjugal transfer of broad-host-range incompatibility group P and Q plasmids from Escherichia coli to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Goncharoff, P; Yip, J K; Wang, H; Schreiner, H C; Pai, J A; Furgang, D; Stevens, R H; Figurski, D H; Fine, D H

    1993-01-01

    The first example of conjugal transfer of DNA from Escherichia coli to the periodontal pathogen Actinobacillus actinomycetemcomitans is presented. Derivatives of the incompatibility group P (IncP) plasmid RK2 successfully transferred from an E. coli donor to an A. actinomycetemcomitans recipient. The resulting A. actinomycetemcomitans transconjugants transferred the plasmids back to E. coli recipients. The IncP transfer functions were also used in trans to mobilize the IncQ plasmid pBK1 from E. coli to A. actinomycetemcomitans. The IncP and IncQ plasmids both transferred into A. actinomycetemcomitans at high frequencies (0.3 to 0.5 transconjugants per donor) and showed no gross deletions, insertions, or rearrangements. Determinations of MICs of various antibiotics for the A. actinomycetemcomitans transconjugant strains demonstrated the expression of ampicillin, chloramphenicol, and kanamycin resistance determinants. Images PMID:8335386

  4. Genetic and Functional Analyses of the Actinobacillus actinomycetemcomitans AfeABCD Siderophore-Independent Iron Acquisition System

    PubMed Central

    Rhodes, Eric R.; Tomaras, Andrew P.; McGillivary, Glen; Connerly, Pamela L.; Actis, Luis A.

    2005-01-01

    The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions. PMID:15908408

  5. Molecular Characterization of an Outer Membrane Protein of Actinobacillus actinomycetemcomitans Belonging to the OmpA Family

    PubMed Central

    White, Peter A.; Nair, Sean P.; Kim, Mi-Jurng; Wilson, Michael; Henderson, Brian

    1998-01-01

    The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34. PMID:9423883

  6. Microarray-based comparative genomic profiling of reference strains and selected Canadian field isolates of Actinobacillus pleuropneumoniae

    PubMed Central

    Gouré, Julien; Findlay, Wendy A; Deslandes, Vincent; Bouevitch, Anne; Foote, Simon J; MacInnes, Janet I; Coulton, James W; Nash, John HE; Jacques, Mario

    2009-01-01

    Background Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines. Results Using hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 A. pleuropneumoniae L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements. Conclusion Although A. pleuropneumoniae strains are essentially clonal, M-CGH analysis of the reference strains of the fifteen serovars and representative field isolates revealed several classes of genes that were divergent or absent. Not surprisingly, these included genes associated with capsule biosynthesis as the capsule is associated with sero-specificity. Several of the conserved genes were identified as candidates for vaccine development, and we conclude that M-CGH is a valuable tool for reverse vaccinology. PMID:19239696

  7. Microbial ecology of Actinobacillus actinomycetemcomitans, Eikenella corrodens and Capnocytophaga spp. in adult periodontitis.

    PubMed

    Müller, H P; Heinecke, A; Borneff, M; Knopf, A; Kiencke, C; Pohl, S

    1997-08-01

    Information on intraoral distribution of putative periodontal pathogens might be essential for controlling different forms of periodontal disease. Colonization may be either promoted or impeded by other bacteria competing in the subgingival ecosystem. In recent investigations microbial associations between dental organisms have been determined in a multitude of subgingival plaque samples within multiple patients and described by odds ratios, in most circumstances without taking into account the correlated structure of the observations within a single individual. The present investigation had 3 major objectives: (i) to describe the intraoral distribution of some facultatively anaerobic, Gram-negative rods, i.e. Actinobacillus actinomycetemcomitans, Eikenella corrodens-like organisms and Capnocytophaga spp., in a multitude of subgingival and extracrevicular samples of 10 adult subjects with A. actinomycetemcomitans-associated periodontitis; (ii) to analyse possible inconsistencies of microbial associations between these periodontal organisms; and (iii) to determine factors increasing the likelihood of isolating these bacteria in a given subgingival site by employing Generalized Estimation Equation (GEE) methods. Clinical examinations were carried out at 6 sites of every tooth present. In each subject, 13 extracrevicular (2 cheek mucosa, 3 tongue, 4 gingival, 2 tonsillar samples, 1 palatinal, 1 saliva sample) and between 22 and 44 subgingival samples from deepest sites of every tooth present (n = 296) were selectively cultivated for A. actinomycetemcomitans, E. corrodens and Capnocytophaga spp. In extracrevicular material, A. actinomycetemcomitans, Capnocytophaga spp. and E. corrodens were isolated in 9, 10 and 6 patients, and from 65, 82 and 15% samples, respectively. The organisms were recovered from 51, 62 and 27% subgingival plaque samples, respectively. Heterogeneity tests did not reveal significant inconsistencies of microbial associations between bacteria in

  8. Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract.

    PubMed

    Valeur, Nana; Engel, Peter; Carbajal, Noris; Connolly, Eamonn; Ladefoged, Karin

    2004-02-01

    Lactobacillus reuteri ATCC 55730 is a probiotic (health-promoting) bacterium widely used as a dietary supplement. This study was designed to examine local colonization of the human gastrointestinal mucosa after dietary supplementation with L. reuteri ATCC 55730 and to determine subsequent immune responses at the colonized sites. In this open clinical investigation, 10 healthy volunteers and 9 volunteers with ileostomy underwent gastroscopy or ileoscopy and biopsy samples were taken from the stomach, duodenum, or ileum before and after supplementation with 4 x 10(8) CFU of live L. reuteri ATCC 55730 lactobacilli per day for 28 days. Biopsy specimen colonization was analyzed using fluorescence in situ hybridization with a molecular beacon probe, and immune cell populations were determined by immunostaining. Endogenous L. reuteri was detected in the stomach of 1 subject and the duodenum of 3 subjects (out of 10 subjects). After L. reuteri ATCC 55730 supplementation, the stomachs of 8 and the duodenums of all 10 subjects were colonized. Three ileostomy subjects (of six tested) had endogenous L. reuteri at baseline, while all six displayed colonization after L. reuteri supplementation. Gastric mucosal histiocyte numbers were reduced and duodenal B-lymphocyte numbers were increased by L. reuteri ATCC 55730 administration. Furthermore, L. reuteri administration induced a significantly higher amount of CD4-positive T-lymphocytes in the ileal epithelium. Dietary supplementation with the probiotic L. reuteri ATCC 55730 induces significant colonization of the stomach, duodenum, and ileum of healthy humans, and this is associated with significant alterations of the immune response in the gastrointestinal mucosa. These responses may be key components of a mechanism by which L. reuteri ATCC 55730 exerts its well-documented probiotic effects in humans.

  9. Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation

    SciTech Connect

    McKeown, Catherine K; Brown, Steven D

    2011-01-01

    The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. A time-series analysis of gene expression revealed changes in transcript levels of {approx}40% of genes ({approx}1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide hydrolysis products

  10. Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation

    PubMed Central

    2011-01-01

    Background The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. Results A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Conclusions Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide

  11. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  12. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H.C.

    1998-07-14

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  13. Fermentation of residual glycerol by Clostridium acetobutylicum ATCC 824 in pure and mixed cultures.

    PubMed

    Dams, Rosemeri I; Guilherme, Alexandre A; Vale, Maria S; Nunes, Vanja F; Leitão, Renato C; Santaella, Sandra T

    2016-12-01

    The aim of this research was to estimate the production of hydrogen, organic acids and alcohols by the strain of Clostridium acetobutylicum ATCC 824 using residual glycerol as a carbon source. The experiments were carried out in pure and mixed cultures in batch experiments. Three different sources of inocula for mixed culture were used. Ruminal liquid from goats and sludge collected from two upflow anaerobic sludge blanket reactors treating municipal wastewater and brewery effluent were tested for hydrogen, organic acids and alcohols production with or without C. acetobutylicum ATCC 824. The main detected end-products from the glycerol fermentation were hydrogen, organic acids (acetic, propionic, butyric and caproic) and alcohol (ethanol and 1,3-propanediol - 1,3PD). High hydrogen (0.44 mol H2/mol glycerol consumed) and 1,3PD (0.32 mol 1,3PD/mol glycerol consumed) yields were obtained when the strain C. acetobutylicum ATCC 824 was bioaugmented into the sludge from municipal wastewater using 5 g/L of glycerol. Significant concentrations of n-caproic acid were detected in the ruminal liquid when amended with C. acetobutylicum ATCC 824. The results suggest that glycerol can be used for the generation of H2, 1,3PD and n-caproic acid using C. acetobutylicum ATCC 824 as agent in pure or mixed cultures.

  14. Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac- to Lac+.

    PubMed

    Tsai, Yu-Kuo; Chen, Hung-Wen; Lo, Ta-Chun; Lin, Thy-Hou

    2009-03-01

    Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac(+) clones could be obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac(-)) and its Lac(+) revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac(+)) strains was high; namely, 96-100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac(+) revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac(+) revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac(-) to Lac(+) for L. casei ATCC 27139. PMID:19246746

  15. Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

    PubMed Central

    Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.

    1998-01-01

    A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. PMID:9501448

  16. Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

    PubMed Central

    Binda, Elisa; Carrano, Lucia; Bibb, Mervyn; Marinelli, Flavia

    2014-01-01

    Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the β-lactam-sensitive d,d-peptidase/d,d-carboxypeptidase encoded by vanYn, the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanYn gene dosage and expressed vanHatAatXat from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanYn, complementing a vanYn mutant, or duplicating vanYn had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYn activity. The heterologous expression of vanHatAatXat increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanYn-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins. PMID:24957828

  17. Relationship between glycopeptide production and resistance in the actinomycete Nonomuraea sp. ATCC 39727.

    PubMed

    Marcone, Giorgia Letizia; Binda, Elisa; Carrano, Lucia; Bibb, Mervyn; Marinelli, Flavia

    2014-09-01

    Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the β-lactam-sensitive D,D-peptidase/D,D-carboxypeptidase encoded by vanYn, the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanYn gene dosage and expressed vanHatAatXat from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanYn, complementing a vanYn mutant, or duplicating vanYn had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYn activity. The heterologous expression of vanHatAatXat increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanYn-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.

  18. CAMP test detected Staphylococcus delphini ATCC 49172 beta-haemolysin production.

    PubMed

    Savini, Vincenzo; Kosecka, Maja; Marrollo, Roberta; Carretto, Edoardo; Miedzobrodzki, Jacek

    2013-01-01

    Through a CAMP test, we first observed a Staphylococcus delphini strain (ATCC 49172) to release beta-haemolysin. Production of the latter in this coagulase-positive species of the 'Staphylococcus intermedius Group', in fact, has been labeled to be undetermined, thus far. Of course, a wider number of strains have to be investigated in order to define whether this property is constitutive (like in Staphylococcus (pseud)intermedius), or strain-dependent (like in Staphylococcus aureus), and which clinical impact it has; nevertheless, we can state that S. delphini ATCC 49172 indeed produces this toxin.

  19. Draft Genome Sequence of Clostridium scatologenes ATCC 25775, a Chemolithoautotrophic Acetogenic Bacterium Producing 3-Methylindole and 4-Methylphenol

    PubMed Central

    Song, Yoseb; Jeong, Yujin; Shin, Hyeon Seok

    2014-01-01

    Clostridium scatologenes ATCC 25775 is a strictly anaerobic and chemolithoautotrophic acetogenic bacterium that converts syngas into multi-carbon compounds such as acetate, indole, 3-methylindole, and 4-methylphenol. Here we report the draft genome sequence of C. scatologenes ATCC 25775 (7.3 Mbp) to elucidate its metabolic pathway for syngas fermentation. PMID:24831152

  20. Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli*

    PubMed Central

    Naegeli, Andreas; Neupert, Christine; Fan, Yao-Yun; Lin, Chia-Wei; Poljak, Kristina; Papini, Anna Maria; Schwarz, Flavio; Aebi, Markus

    2014-01-01

    N-Linked protein glycosylation is a frequent post-translational modification that can be found in all three domains of life. In a canonical, highly conserved pathway, an oligosaccharide is transferred by a membrane-bound oligosaccharyltransferase from a lipid donor to asparagines in the sequon NX(S/T) of secreted polypeptides. The δ-proteobacterium Actinobacillus pleuropneumoniae encodes an unusual pathway for N-linked protein glycosylation. This pathway takes place in the cytoplasm and is mediated by a soluble N-glycosyltransferase (NGT) that uses nucleotide-activated monosaccharides to glycosylate asparagine residues. To characterize the process of cytoplasmic N-glycosylation in more detail, we studied the glycosylation in A. pleuropneumoniae and functionally transferred the glycosylation system to Escherichia coli. N-Linked glucose specific human sera were used for the analysis of the glycosylation process. We identified autotransporter adhesins as the preferred protein substrate of NGT in vivo, and in depth analysis of the modified sites in E. coli revealed a surprisingly relaxed peptide substrate specificity. Although NX(S/T) is the preferred acceptor sequon, we detected glycosylation of alternative sequons, including modification of glutamine and serine residues. We also demonstrate the use of NGT to glycosylate heterologous proteins. Therefore, our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria. PMID:24275653

  1. ICEApl1, an Integrative Conjugative Element Related to ICEHin1056, Identified in the Pig Pathogen Actinobacillus pleuropneumoniae.

    PubMed

    Bossé, Janine T; Li, Yanwen; Fernandez Crespo, Roberto; Chaudhuri, Roy R; Rogers, Jon; Holden, Matthew T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2016-01-01

    ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia. PMID:27379024

  2. Changes in gene expression of Actinobacillus pleuropneumoniae in response to anaerobic stress reveal induction of central metabolism and biofilm formation.

    PubMed

    Li, Lu; Zhu, Jiawen; Yang, Kui; Xu, Zhuofei; Liu, Ziduo; Zhou, Rui

    2014-06-01

    Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry worldwide. Oxygen deprivation is a stress that A. pleuropneumoniae will encounter during both early infection and the later, persistent stage. To understand modulation of A. pleuropneumoniae gene expression in response to the stress caused by anaerobic conditions, gene expression profiles under anaerobic and aerobic conditions were compared in this study. The microarray results showed that 631 genes (27.7% of the total ORFs) were differentially expressed in anaerobic conditions. Many genes encoding proteins involved in glycolysis, carbon source uptake systems, pyruvate metabolism, fermentation and the electron respiration transport chain were up-regulated. These changes led to an increased amount of pyruvate, lactate, ethanol and acetate in the bacterial cells as confirmed by metabolite detection. Genes encoding proteins involved in cell surface structures, especially biofilm formation, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis were up-regulated as well. Biofilm formation was significantly enhanced under anaerobic conditions. These results indicate that induction of central metabolism is important for basic survival of A. pleuropneumoniae after a shift to an anaerobic environment. Enhanced biofilm formation may contribute to the persistence of this pathogen in the damaged anaerobic host tissue and also in the early colonization stage. These discoveries give new insights into adaptation mechanisms of A. pleuropneumoniae in response to environmental stress.

  3. Viability of Actinobacillus pleuropneumoniae in frozen pig lung samples and comparison of different methods of direct diagnosis in fresh samples.

    PubMed

    Gutierrez, C B; Rodriguez Barbosa, J I; Gonzalez, O R; Tascon, R I; Rodriguez Ferri, E F

    1992-04-01

    A comparative study on different methods of diagnosis of Actinobacillus pleuropneumoniae from both fresh and frozen pig lungs is described. A total of 196 lung tissues with pneumonic lesions were examined for culture isolation on chocolate blood agar, as well as for antigen detection by means of the coagglutination test, the immunodiffusion test and the indirect ELISA. These samples were subsequently frozen for 1 yr and then they were recultured. A. pleuropneumoniae was recovered from fresh lung specimens in 30 cases (15.3%) and from frozen samples in only two cases (0.9%). Such a different degree of isolation demonstrates that long freezing had an adverse effect on the viability of this organism in lung samples. A pleuropneumoniae detection was positive in 134 samples (68.4%) by at least one of the immunological techniques examined. The indirect ELISA was the most sensitive and specific test, with antigen detected in 125 lungs (63.8%). In comparison with the coagglutination and immunodiffusion tests, the sensitivities of the indirect ELISA were 95.8 and 93.7%, and the specificities were 67.0 and 63.4%, respectively.

  4. Immunoprotective Efficacy of Six In vivo-Induced Antigens against Actinobacillus pleuropneumoniae as Potential Vaccine Candidates in Murine Model

    PubMed Central

    Zhang, Fei; Cao, Sanjie; Zhu, Zhuang; Yang, Yusheng; Wen, Xintian; Chang, Yung-Fu; Huang, Xiaobo; Wu, Rui; Wen, Yiping; Yan, Qigui; Huang, Yong; Ma, Xiaoping; Zhao, Qin

    2016-01-01

    Six in vivo-induced (IVI) antigens—RnhB, GalU, GalT, Apl_1061, Apl_1166, and HflX were selected for a vaccine trial in a mouse model. The results showed that the IgG levels in each immune group was significantly higher than that of the negative control (P < 0.001). Except rRnhB group, proliferation of splenocytes was observed in all immunized groups and a relatively higher proliferation activity was observed in rGalU and rGalT groups (P < 0.05). In the rGalT vaccinated group, the proportion of CD4+ T cells in spleen was significant higher than that of negative control (P < 0.05). Moreover, proportions of CD4+ T cells in other vaccinated groups were all up-regulated to varying degrees. Up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokines were detected. A survival rate of 87.5, 62.5, and 62.5% were obtained among rGalT, rAPL_1166, and rHflX group, respectively while the remaining three groups was only 25%. Histopathological analyses of lungs indicated that surviving animals from the vaccinated groups showed relatively normal pulmonary structure alveoli. These findings confirm that IVI antigens used as vaccine candidates provide partial protection against Actinobacillus pleuropneumoniae infection in a mouse model, which could be used as potential vaccine candidates in piglets.

  5. 16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains.

    PubMed

    Ihalin, Riikka; Asikainen, Sirkka

    2006-06-01

    Certain serotypes of Actinobacillus actinomycetemcomitans seem to prefer coexistence in vivo. The 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was tested for its capability to distinguish coexisting A. actinomycetemcomitans strains of different serotypes or genetic lineages and to determine their proportions in vitro. The migration pattern of the PCR amplicon from serotype c differed from those of the other serotypes. Contrary to the strains of serotypes c, d, and e, strains of serotypes a, b, and f consistently demonstrated intra-serotype migration patterns similar to each other. Since the migration patterns differed between serotype c and b strains a strain of each was used to determine their proportional representation in a strain mixture. The strains were distinguishable from each other above the 5% PCR-DGGE detection level (12.5 ng DNA/1.5 x 10(6) cells). DGGE provides a promising tool for in vitro studies on the coexistence of different genetic lineages of A. actinomycetemcomitans.

  6. Susceptibility testing of Actinobacillus pleuropneumoniae in Denmark. Evaluation of three different media of MIC-determinations and tablet diffusion tests.

    PubMed

    Aarestrup, F M; Jensen, N E

    1999-02-12

    This study was conducted to compare the applicability of three different media in sensitivity testing of Actinobacillus pleuropneumoniae by means of MIC and tablet diffusion tests. The media used were: modified PPLO agar, chocolatized Mueller-Hinton-II and Columbia agar supplemented with NAD. Seven antimicrobial agents were tested: ceftiofur, enrofloxacin, penicillin, spectinomycin, tiamulin, trimethoprim + sulfadiazine and tylosin, against 40 randomly selected A. pleuropneumoniae isolates. In general, good agreement was found between results obtained with all combinations of media, most antimicrobials tested and the two-test systems. Some variations between media were observed for spectinomycin, tiamulin and tylosin. For ceftiofur and trimethoprim + sulfadiazine some isolates with low MIC-values were classified as resistant using tablet diffusion, indicating that the break points of resistance for these antimicrobials using the tablet diffusion tests need adjustment. Using current break points for resistance with MIC-determinations, all isolates tested susceptible to ceftiofur, enrofloxacin, penicillin, tiamulin and trimethoprim + sulfadiazine. A larger number of isolates tested resistant to spectinomycin and tylosin on all three media using both MIC determinations and tablet diffusion. PMID:10063535

  7. Genomic relatedness among Actinobacillus pleuropneumoniae field strains of sterotypes 1 and 5 isolated from healthy and diseased pigs.

    PubMed Central

    Chatellier, S; Harel, J; Dugourd, D; Chevallier, B; Kobisch, M; Gottschalk, M

    1999-01-01

    Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the molecular epidemiology of swine pleuropneumonia. Images Figure 1. Figure 3. PMID:10480458

  8. Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.

    PubMed Central

    Ward, C K; Inzana, T J

    1997-01-01

    Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens. PMID:9169799

  9. ICEApl1, an Integrative Conjugative Element Related to ICEHin1056, Identified in the Pig Pathogen Actinobacillus pleuropneumoniae

    PubMed Central

    Bossé, Janine T.; Li, Yanwen; Fernandez Crespo, Roberto; Chaudhuri, Roy R.; Rogers, Jon; Holden, Matthew T. G.; Maskell, Duncan J.; Tucker, Alexander W.; Wren, Brendan W.; Rycroft, Andrew N.; Langford, Paul R.

    2016-01-01

    ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia. PMID:27379024

  10. Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

    PubMed Central

    Yamaguchi, N; Yamashita, Y; Ikeda, D; Koga, T

    1996-01-01

    Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it. Human monocytes stimulated with SPA exhibited the increased mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and a neutrophil chemotactic factor, interleukin-8 (IL-8). On the other hand, SPA induced the release of IL-1, IL-6, and tumor necrosis factor (TNF) and enhanced the expression of IL-1alpha, IL-1beta, IL-6, and TNF alpha (TNF-alpha) mRNAs. Human monocytes expressed MCP-1 and IL-8 mRNAs when stimulated by human recombinant IL-1alpha, I1-1beta, IL-6, and TNF-alpha, suggesting that these inflammatory cytokines induced by SPA might participate in the production of chemotactic factors in human monocytes. PMID:8698480

  11. Characterization of the omlA gene from different serotypes of Actinobacillus pleuropneumoniae: A new insight into an old approach

    PubMed Central

    Rossi, Ciro César; de Araújo, Elza Fernandes; de Queiroz, Marisa Vieira; Bazzolli, Denise Mara Soares

    2013-01-01

    The OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein. PMID:23885207

  12. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582

    NASA Astrophysics Data System (ADS)

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S.; Ellis, Tom

    2016-03-01

    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.

  13. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

    PubMed

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S; Ellis, Tom

    2016-01-01

    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. PMID:27010592

  14. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582

    PubMed Central

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S.; Ellis, Tom

    2016-01-01

    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. PMID:27010592

  15. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

    PubMed

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S; Ellis, Tom

    2016-03-24

    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.

  16. Complete Genome Sequence of the Larval Shellfish Pathogen Vibrio tubiashii Type Strain ATCC 19109.

    PubMed

    Richards, Gary P; Needleman, David S; Watson, Michael A; Bono, James L

    2014-12-18

    Vibrio tubiashii is a larval shellfish pathogen. Here, we report the first closed genome sequence for this species (ATCC type strain 19109), which consists of two chromosomes (3,294,490 and 1,766,582 bp), two megaplasmids (251,408 and 122,808 bp), and two plasmids (57,076 and 47,973 bp).

  17. Complete Genome Sequence of Thiostrepton-Producing Streptomyces laurentii ATCC 31255

    PubMed Central

    Fujino, Yasuhiro; Nagayoshi, Yuko; Ohshima, Toshihisa; Ogata, Seiya

    2016-01-01

    Streptomyces laurentii ATCC 31255 produces thiostrepton, a thiopeptide class antibiotic. Here, we report the complete genome sequence for this strain, which contains a total of 8,032,664 bp, 7,452 predicted coding sequences, and a G+C content of 72.3%. PMID:27257211

  18. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

    PubMed Central

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-01-01

    Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain. PMID:27305971

  19. Complete Genome Sequence of Brachyspira hyodysenteriae Type Strain B-78 (ATCC 27164).

    PubMed

    Mirajkar, Nandita S; Johnson, Timothy J; Gebhart, Connie J

    2016-01-01

    Reported herein is the complete genome sequence of the type strain B-78 (ATCC 27164) of Brachyspira hyodysenteriae, the etiological agent of swine dysentery. The 3.1-Mb genome consists of a 3.056-Mb chromosome and a 45-kb plasmid, with 2,617 protein-coding genes, 39 RNA genes, and 40 pseudogenes. PMID:27540064

  20. Finished Genome Sequence of the Laboratory Strain Escherichia coli K-12 RV308 (ATCC 31608).

    PubMed

    Krempl, Peter M; Mairhofer, Juergen; Striedner, Gerald; Thallinger, Gerhard G

    2014-11-20

    Escherichia coli strain K-12 substrain RV308 is an engineered descendant of the K-12 wild-type strain. Like its ancestor, it is an important organism in biotechnological research and is heavily used for the expression of single-chain variable fragments. Here, we report the complete genome sequence of E. coli K-12 RV308 (ATCC 31608).

  1. Finished Genome Sequence of Escherichia coli K-12 Strain HMS174 (ATCC 47011).

    PubMed

    Mairhofer, Juergen; Krempl, Peter M; Thallinger, Gerhard G; Striedner, Gerald

    2014-11-20

    Escherichia coli strain K-12 substrain HMS174 is an engineered descendant of the E. coli K-12 wild-type strain. Like its ancestor, it is an important organism in biotechnological research and is used in fermentation processes for heterologous protein production. Here, we report the complete genome sequence of E. coli HMS174 (ATCC 47011).

  2. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock

    PubMed Central

    Chang, Philip Lee; Yen, Teh Fu

    1985-01-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed. PMID:16346956

  3. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock.

    PubMed

    Chang, P L; Yen, T F

    1985-12-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed.

  4. Properties of a hydrogen-inhibited mutant of Desulfovibrio desulfuricans ATCC 27774.

    PubMed Central

    Odom, J M; Wall, J D

    1987-01-01

    A mutant of Desulfovibrio desulfuricans ATCC 27774 has been obtained which is incapable of sulfate respiration with molecular hydrogen but which grows normally on lactate plus sulfate under argon. Growth characteristics of the mutant suggest that the defect is involved in electron transfer to sulfate or nitrate but not thiosulfate. PMID:3818548

  5. Complete Genome Sequence of Thiostrepton-Producing Streptomyces laurentii ATCC 31255.

    PubMed

    Doi, Katsumi; Fujino, Yasuhiro; Nagayoshi, Yuko; Ohshima, Toshihisa; Ogata, Seiya

    2016-06-02

    Streptomyces laurentii ATCC 31255 produces thiostrepton, a thiopeptide class antibiotic. Here, we report the complete genome sequence for this strain, which contains a total of 8,032,664 bp, 7,452 predicted coding sequences, and a G+C content of 72.3%.

  6. Complete Genome Sequence and Methylome Analysis of Bacillus globigii ATCC 49760.

    PubMed

    Morgan, Richard D

    2016-05-26

    Bacillus subtilis (Ehrenburg) Cohn ATCC 49760, deposited as Bacillus globigii, is the source strain for the restriction enzymes BglI and BglII. Its complete sequence and full methylome were determined using single-molecule real-time (SMRT) sequencing.

  7. Genome Sequence of Streptomyces viridosporus Strain T7A ATCC 39115, a Lignin-Degrading Actinomycete

    SciTech Connect

    Davis, Jennifer R.; Goodwin, Lynne A.; Teshima, Hazuki; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Huntemann, Marcel; Wei, Chia-Lin; Han, James; Chen, Amy; Kyrpides, Nikos C; Mavromatis, K; Szeto, Ernest; Markowitz, Victor; Ivanova, N; Mikhailova, Natalia; Ovchinnikova, Galina; Pagani, Ioanna; Pati, Amrita; Woyke, Tanja; Pitluck, Sam; Peters, Lin; Nolan, Matt; Land, Miriam L; Sello, Jason K.

    2013-01-01

    We announce the availability of the genome sequence of Streptomyces viridosporus strain T7A ATCC 39115, a plant biomass- degrading actinomycete. This bacterium is of special interest because of its capacity to degrade lignin, an underutilized compo- nent of plants in the context of bioenergy. It has a full complement of genes for plant biomass catabolism.

  8. Draft Genome Assembly of Bordetella bronchiseptica ATCC 10580, a Historical Canine Clinical Isolate.

    PubMed

    Daligault, H E; Davenport, K W; Minogue, T D; Bishop-Lilly, K A; Bruce, D C; Chain, P S; Coyne, S R; Frey, K G; Jaissle, J; Koroleva, G I; Ladner, J T; Lo, C-C; Meincke, L; Munk, C; Palacios, G F; Redden, C L; Johnson, S L

    2014-01-01

    We present the scaffolded genome of Bordetella bronchiseptica ATCC 10580, assembled into 98 contigs. This 5.1-Mb assembly (68.2% G+C content) contains 4,870 coding regions. The strain was originally isolated from canine lung tissue and is used in quality control testing. PMID:25237025

  9. Genome Assembly of Shigella flexneri ATCC 12022, a Quality Control Reference Strain

    PubMed Central

    Daligault, H. E.; Davenport, K. W.; Minogue, T. D.; Bishop-Lilly, K. A.; Broomall, S. M.; Bruce, D. C.; Chain, P. S.; Coyne, S. R.; Freitas, T.; Frey, K. G.; Gibbons, H. S.; Jaissle, J.; Lo, C.-C.; Meincke, L.; Munk, A. C.; Redden, C. L.; Rosenzweig, C. N.

    2014-01-01

    Shigella flexneri causes shigellosis, severe and potentially life-threatening diarrhea, and accounts for 18% of shigellosis cases in the United States. Here, we present the 4.51-Mbp genome assembly of S. flexneri ATCC 12022, a quality control and reference strain, in 10 scaffolds. PMID:25359907

  10. Complete Genome Sequence of the Quality Control Strain Staphylococcus aureus subsp. aureus ATCC 25923

    PubMed Central

    Treangen, Todd J.; Maybank, Rosslyn A.; Enke, Sana; Friss, Mary Beth; Diviak, Lynn F.; Karaolis, David K. R.; Koren, Sergey; Ondov, Brian; Phillippy, Adam M.; Bergman, Nicholas H.

    2014-01-01

    Staphylococcus aureus subsp. aureus ATCC 25923 is commonly used as a control strain for susceptibility testing to antibiotics and as a quality control strain for commercial products. We present the completed genome sequence for the strain, consisting of the chromosome and a 27.5-kb plasmid. PMID:25377701

  11. Complete Genome Sequence of the Quality Control Strain Staphylococcus aureus subsp. aureus ATCC 25923.

    PubMed

    Treangen, Todd J; Maybank, Rosslyn A; Enke, Sana; Friss, Mary Beth; Diviak, Lynn F; Karaolis, David K R; Koren, Sergey; Ondov, Brian; Phillippy, Adam M; Bergman, Nicholas H; Rosovitz, M J

    2014-01-01

    Staphylococcus aureus subsp. aureus ATCC 25923 is commonly used as a control strain for susceptibility testing to antibiotics and as a quality control strain for commercial products. We present the completed genome sequence for the strain, consisting of the chromosome and a 27.5-kb plasmid. PMID:25377701

  12. Complete Genome Sequence of Brachyspira hyodysenteriae Type Strain B-78 (ATCC 27164)

    PubMed Central

    Mirajkar, Nandita S.; Johnson, Timothy J.

    2016-01-01

    Reported herein is the complete genome sequence of the type strain B-78 (ATCC 27164) of Brachyspira hyodysenteriae, the etiological agent of swine dysentery. The 3.1-Mb genome consists of a 3.056-Mb chromosome and a 45-kb plasmid, with 2,617 protein-coding genes, 39 RNA genes, and 40 pseudogenes. PMID:27540064

  13. Effect of Calcium in Assay Medium on D Value of Bacillus stearothermophilus ATCC 7953 Spores

    PubMed Central

    Sasaki, Koichi; Shintani, Hideharu; Itoh, Junpei; Kamogawa, Takuji; Kajihara, Yousei

    2000-01-01

    The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value. PMID:11097939

  14. Draft Genome Sequence of the Probiotic Strain Lactobacillus acidophilus ATCC 4356

    PubMed Central

    Palomino, Maria Mercedes; Allievi, Mariana C.; Fina Martin, Joaquina; Waehner, Pablo M.; Prado Acosta, Mariano; Sanchez Rivas, Carmen

    2015-01-01

    We present the 1,956,699-bp draft genome sequence of Lactobacillus acidophilus strain ATCC 4356. Comparative genomic analysis revealed 99.96% similarity with L. acidophilus NCFM NC_006814.3 and 99.97% with La-14 NC_021181.2 genomes. PMID:25593259

  15. Antimicrobial mechanism of flavonoids against Escherichia coli ATCC 25922 by model membrane study

    NASA Astrophysics Data System (ADS)

    He, Mengying; Wu, Ting; Pan, Siyi; Xu, Xiaoyun

    2014-06-01

    Antimicrobial mechanism of four flavonoids (kaempferol, hesperitin, (+)-catechin hydrate, biochanin A) against Escherichia coli ATCC 25922 was investigated through cell membranes and a liposome model. The release of bacterial protein and images from transmission electron microscopy demonstrated damage to the E. coli ATCC 25922 membrane. A liposome model with dipalmitoylphosphatidylethanolamine (DPPE) (0.6 molar ratio) and dipalmitoylphosphatidylglycerol (DPPG) (0.4 molar ratio), representative of the phospholipid membrane of E. coli ATCC 25922, was used to specify the mode of action of four selected flavonoids through Raman spectroscopy and differential scanning calorimetry. It is suggested that for flavonoids, to be effective antimicrobials, interaction with the polar head-group of the model membrane followed by penetration into the hydrophobic regions must occur. The antimicrobial efficacies of the flavonoids were consistent with liposome interaction activities, kaempferol > hesperitin > (+)-catechin hydrate > biochanin A. This study provides a liposome model capable of mimicking the cell membrane of E. coli ATCC 25922. The findings are important in understanding the antibacterial mechanism on cell membranes.

  16. Complete genome sequence of the beer spoilage organism Pediococcus claussenii ATCC BAA-344T.

    PubMed

    Pittet, Vanessa; Abegunde, Teju; Marfleet, Travis; Haakensen, Monique; Morrow, Kendra; Jayaprakash, Teenus; Schroeder, Kristen; Trost, Brett; Byrns, Sydney; Bergsveinson, Jordyn; Kusalik, Anthony; Ziola, Barry

    2012-03-01

    Pediococcus claussenii is a common brewery contaminant. We have sequenced the chromosome and plasmids of the type strain P. claussenii ATCC BAA-344. A ropy variant was chosen for sequencing to obtain genetic information related to growth in beer, as well as exopolysaccharide and possibly biofilm formation by this organism. PMID:22328764

  17. Draft Genome Sequence of Streptomyces silvensis ATCC 53525, a Producer of Novel Hormone Antagonists

    PubMed Central

    Johnston, Chad W.; Li, Yongchang

    2016-01-01

    Streptomyces silvensis produces nonribosomal peptides that act as antagonists of the human oxytocin and vasopressin receptors. Here, we present the genome sequence of S. silvensis ATCC 53525 and demonstrate that this organism possesses a number of additional biosynthetic gene clusters and might be a promising source for genome-guided drug discovery efforts. PMID:26893408

  18. Draft Genome Assembly of Bordetella bronchiseptica ATCC 10580, a Historical Canine Clinical Isolate.

    PubMed

    Daligault, H E; Davenport, K W; Minogue, T D; Bishop-Lilly, K A; Bruce, D C; Chain, P S; Coyne, S R; Frey, K G; Jaissle, J; Koroleva, G I; Ladner, J T; Lo, C-C; Meincke, L; Munk, C; Palacios, G F; Redden, C L; Johnson, S L

    2014-01-01

    We present the scaffolded genome of Bordetella bronchiseptica ATCC 10580, assembled into 98 contigs. This 5.1-Mb assembly (68.2% G+C content) contains 4,870 coding regions. The strain was originally isolated from canine lung tissue and is used in quality control testing.

  19. Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans.

    PubMed

    Helgeland, K; Nordby, O

    1993-05-01

    A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired. PMID:8496779

  20. Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579.

    PubMed

    Tagawa, Yuichi

    2014-12-01

    Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.

  1. Identification of Campylobacter jejuni ATCC 43431-Specific Genes by Whole Microbial Genome Comparisons

    PubMed Central

    Poly, Frédéric; Threadgill, Deborah; Stintzi, Alain

    2004-01-01

    This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis. PMID:15231810

  2. Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-08-04

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials. 5 figs.

  3. Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials.

  4. Lactobacillus acidophilus ATCC 4356 prevents atherosclerosis via inhibition of intestinal cholesterol absorption in apolipoprotein E-knockout mice.

    PubMed

    Huang, Ying; Wang, Jinfeng; Quan, Guihua; Wang, Xiaojun; Yang, Longfei; Zhong, Lili

    2014-12-01

    The objective of this study was to investigate the effect of Lactobacillus acidophilus ATCC 4356 on the development of atherosclerosis in apolipoprotein E-knockout (ApoE(-/-)) mice. Eight-week-old ApoE(-/-) mice were fed a Western diet with or without L. acidophilus ATCC 4356 daily for 16 weeks. L. acidophilus ATCC 4356 protected ApoE(-/-) mice from atherosclerosis by reducing their plasma cholesterol levels from 923 ± 44 to 581 ± 18 mg/dl, likely via a marked decrease in cholesterol absorption caused by modulation of Niemann-Pick C1-like 1 (NPC1L1). In addition, suppression of cholesterol absorption induced reverse cholesterol transport (RCT) in macrophages through the peroxisome proliferator-activated receptor/liver X receptor (PPAR/LXR) pathway. Fecal lactobacillus and bifidobacterium counts were significantly (P < 0.05) higher in the L. acidophilus ATCC 4356 treatment groups than in the control groups. Furthermore, L. acidophilus ATCC 4356 was detected in the rat small intestine, colon, and feces during the feeding trial. The bacterial levels remained high even after the administration of lactic acid bacteria had been stopped for 2 weeks. These results suggest that administration of L. acidophilus ATCC 4356 can protect against atherosclerosis through the inhibition of intestinal cholesterol absorption. Therefore, L. acidophilus ATCC 4356 may be a potential therapeutic material for preventing the progression of atherosclerosis.

  5. Stability of free and encapsulated Lactobacillus acidophilus ATCC 4356 in yogurt and in an artificial human gastric digestion system.

    PubMed

    Ortakci, F; Sert, S

    2012-12-01

    The objective of this study was to determine the effect of encapsulation on survival of probiotic Lactobacillus acidophilus ATCC 4356 (ATCC 4356) in yogurt and during artificial gastric digestion. Strain ATCC 4356 was added to yogurt either encapsulated in calcium alginate or in free form (unencapsulated) at levels of 8.26 and 9.47 log cfu/g, respectively, and the influence of alginate capsules (1.5 to 2.5mm) on the sensorial characteristics of yogurts was investigated. The ATCC 4356 strain was introduced into an artificial gastric solution consisting of 0.08 N HCl (pH 1.5) containing 0.2% NaCl or into artificial bile juice consisting of 1.2% bile salts in de Man, Rogosa, and Sharpe broth to determine the stability of the probiotic bacteria. When incubated for 2h in artificial gastric juice, the free ATCC 4356 did not survive (reduction of >7 log cfu/g). We observed, however, greater survival of encapsulated ATCC 4356, with a reduction of only 3 log cfu/g. Incubation in artificial bile juice (6 h) did not significantly affect the viability of free or encapsulated ATCC 4356. Moreover, statistically significant reductions (~1 log cfu/g) of both free and encapsulated ATCC 4356 were observed during 4-wk refrigerated storage of yogurts. The addition of probiotic cultures in free or alginate-encapsulated form did not significantly affect appearance/color or flavor/odor of the yogurts. However, significant deficiencies were found in body/texture of yogurts containing encapsulated ATCC 4356. We concluded that incorporation of free and encapsulated probiotic bacteria did not substantially change the overall sensory properties of yogurts, and encapsulation in alginate using the extrusion method greatly enhanced the survival of probiotic bacteria against an artificial human gastric digestive system.

  6. Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaccinated pigs.

    PubMed Central

    Furesz, S E; Mallard, B A; Bossé, J T; Rosendal, S; Wilkie, B N; MacInnes, J I

    1997-01-01

    Current porcine pleuropneumonia bacterins afford only partial protection by decreasing mortality but not morbidity. In order to better understand the type(s) of immune response associated with protection, antibody- and cell-mediated immune responses (CMIR) were compared for piglets before and after administration of a commercial bacterin, which confers partial protection, or a low-dose (10(5) CFU/ml) aerosol challenge with Actinobacillus pleuropneumoniae CM5 (LD), which induces complete protection. Control groups received phosphate-buffered saline or adjuvant. Serum antibody response, antibody avidity, delayed-type hypersensitivity (DTH), and lymphocyte blastogenic responses were measured and compared among treatment groups to the lipopolysaccharide (LPS), capsular polysaccharide (CPS), hemolysin (HLY), and outer membrane proteins (OMP) of A. pleuropneumoniae. Peripheral blood lymphocytes and sera were collected prior to and following primary and secondary immunization-infection and high-dose A. pleuropneumoniae CM5 (10(7) CFU/ml) aerosol challenge. Serum antibody and DTH, particularly that to HLY, differed significantly between treatment groups, and increases were associated with protection. LD-infected piglets had higher antibody responses (P < or = 0.01) and antibody avidity (P < or = 0.10) than bacterin-vaccinated and control groups. Anti-HLY antibodies were consistently associated with protection, whereas anti-LPS and anti-CPS antibodies were not. LD-infected animals had higher DTH responses, particularly to HLY, than bacterin-vaccinated pigs (P < or = 0.03). The LD-infected group maintained consistent blastogenic responses to HLY, LPS, CPS, and OMP over the course of infection, unlike the bacterin-vaccinated and control animals. These data suggest that the immune responses induced by a commercial bacterin are very different from those induced by LD aerosol infection and that current bacterins may be modified, for instance, by addition of HLY, so as to

  7. The genetic organisation of the capsule biosynthesis region of Actinobacillus pleuropneumoniae serotypes 1, 6, 7, and 12.

    PubMed

    Jessing, Stine G; Ahrens, Peter; Inzana, Thomas J; Angen, Øystein

    2008-06-22

    The aim of the present study was to investigate the organisation of the genes (cps) involved in biosynthesis the capsular polysaccharide (CPS) of Actinobacillus pleuropneumoniae serotypes 6, 7, and 12 and to compare these to the corresponding genes previously described in other A. pleuropneumoniae serotypes. In serotypes 6 and 7 the sequenced DNA regions comprised five and four open reading frames, respectively, designated cps6ABCDE and cps7ABCD, whereas the sequenced DNA region in serotype 12 comprised only two open reading frames designated cps12AB. At the amino acid level, CpsA, CpsB, and CpsC of A. pleuropneumoniae serotypes 2, 6, 7, and 8 contained a high degree of homology. At the amino acid level Cps6D revealed a high degree of homology to Cps8D, whereas Cps7D contained a high degree of homology to the Cps2D. The deduced gene product of the partially sequenced cps6E gene showed no homology to any deduced gene products of any cps genes of A. pleuropneumoniae investigated so far. None of the deduced gene products of the cps genes involved in encapsulation of A. pleuropneumoniae serotypes 2, 6, 7, and 8 revealed homology to the deduced gene products of the cps genes of serotypes 1, 5A, and 12. For some genes, a local homology was found to genes probably involved in teichoic acid synthesis in other bacteria. The results obtained revealed a high degree of homology among the genes involved in CPS biosynthesis for serotypes 2, 6, 7, and 8 and a different group of homologous cps genes for serotypes 1 and 12. In some serotype 7 strains, including the serotype 7 reference strain, WF83, the cps genes were not located adjacent to the genes responsible for CPS export (cpx), probably due to genetic rearrangements.

  8. Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

    PubMed Central

    Trottier, Y L; Wright, P F; Larivière, S

    1992-01-01

    An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5. PMID:1734068

  9. Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

    PubMed Central

    Negrete-Abascal, E; Tenorio, V R; Guerrero, A L; García, R M; Reyes, M E; de la Garza, M

    1998-01-01

    A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae. Images Figure 2A. Figure 2B. Figure 3. Figure 4. Figure 5A. Figure 5B. Figure 6A. Figure 6B. PMID:9684047

  10. Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis.

    PubMed Central

    Choi, J I; Ha, M H; Kim, J H; Kim, S J

    1996-01-01

    The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP). PMID:8926092

  11. Comparison of four lung scoring systems for the assessment of the pathological outcomes derived from Actinobacillus pleuropneumoniae experimental infections

    PubMed Central

    2014-01-01

    Background In this study, four lung lesion scoring methods (Slaughterhouse Pleurisy Evaluation System [SPES], Consolidation Lung Lesion Score [LLS], Image analyses [IA] and Ratio of lung weight/body weight [LW/BW]) were compared for the assessment of the different pathological outcomes derived from an Actinobacillus pleuropneumoniae (App) experimental infection model. Moreover, pathological data was coupled with clinical (fever, inappetence and clinical score), production (average daily weigh gain [ADWG]) and diagnostic (PCR, ELISA and bacterial isolation) parameters within the four infection outcomes (peracute, acute, subclinically infected and non-infected). Results From the 61 inoculated animals, 9 were classified as peracute (presence of severe App-like clinical signs and lesions and sudden death or euthanasia shortly after inoculation), 31 as acutely affected (presence of App-like clinical signs and lesions and survival until the end of the experiment), 12 as subclinically infected (very mild or no clinical signs but App infection confirmed) and 9 as non-infected animals (lack of App-like clinical signs and lack of evidence of App infection). A significant correlation between all lung lesion scoring systems was found with the exception of SPES score versus LW/BW. SPES showed a statistically significant association with all clinical, production and diagnostic (with the exception of PCR detection of App in the tonsil) variables assessed. LLS and IA showed similar statistically significant associations as SPES, with the exception of seroconversion against App at necropsy. In contrast, LW/BW was statistically associated only with App isolation in lungs, presence of App-like lesions and ELISA OD values at necropsy. Conclusions In conclusion, SPES, LLS and IA are economic, fast and easy-to-perform lung scoring methods that, in combination with different clinical and diagnostic parameters, allow the characterization of different outcomes after App infection. PMID

  12. Oral immunization against porcine pleuropneumonia using the cubic phase of monoolein and purified toxins of Actinobacillus pleuropneumoniae.

    PubMed

    Lopez-Bermudez, Jorge; Quintanar-Guerrero, David; Lara Puente, Horacio; Tórtora Perez, Jorge; Suárez Güemez, Francisco; Ciprián Carrasco, Abel; Mendoza Elvira, Susana

    2014-11-28

    The main goal of this work was to obtain an orally administered immunogen that would protect against infections by Actinobacillus pleuropneumoniae. The Apx I, II and III toxins were obtained from the supernatants of cultures of serotypes 1 and 3 of A. pleuropneumoniae. The capacity of monoolein gel to trap and protect the Apx toxins, and the effect of their incorporation on the stability of the cubic phase were evaluated. The gel was capable of trapping a 400-μg/ml concentration of the antigen with no effects on its structure. Approximately 60% of the protein molecules were released from the gel within 4h. Four experimental groups were formed, each one with four pigs. All challenges were conducted in a nebulization chamber. Group A: Control (-) not vaccinated and not challenged; Group B: Control (+) not vaccinated but challenged; Group C: vaccinated twice intramuscularly with ToxCom (a commercial toxoid) at an interval of 15 days and then challenged; and Group D: vaccinated orally twice a week for 4 weeks with ToxOral (an oral toxoid) and challenged on day 28 of the experiment with a same dose of 2.0 × 10(4) UFC of A. pleuropneumoniae serotypes 1 and 3. The lesions found in group B covered 27.7-43.1% of the lungs; the pigs in group C had lesions over 12.3-28%; and those in group D over 15.4-32.3%. No lesions were found in the Group A pigs. A. pleuropneumoniae induced macroscopic lesions characteristic of infection by and lesions microscopic detected by histopathology. The etiologic agent was recovered from the infected lungs, tonsils and spleen. The serotypes identified were 1 and 3. An indirect ELISA test identified the antibodies against the Apx toxins in the serum of the animals immunized orally.

  13. Porcine mononuclear phagocyte subpopulations in the lung, blood and bone marrow: dynamics during inflammation induced by Actinobacillus pleuropneumoniae

    PubMed Central

    Ondrackova, Petra; Nechvatalova, Katerina; Kucerova, Zdenka; Leva, Lenka; Dominguez, Javier; Faldyna, Martin

    2010-01-01

    Mononuclear phagocytes (MP) are cells of nonspecific immunity, playing an essential role in defense against bacterial pathogens. Although various MP subpopulations have been described in the pig, relations among these populations in vivo are unknown to date. The present study was aimed at describing porcine MP subpopulations infiltrating inflamed tissue of pigs under in vivo conditions. Actinobacillus pleuropneumoniae (APP) infection was used to induce an inflammatory response. CD172α, CD14, CD163, MHCII and CD203α cell surface molecules were used to identify MP by flow cytometry. Changes in MP subpopulations in the peripheral blood (PB) and bone marrow (BM) compartments along with the analysis of MP appearing in the inflamed lungs were assessed to elucidate the possible origin and maturation stages of the infiltrating MP. The MP population migrating to the inflamed lungs was phenotype CD14+ CD163+ CD203α+/− MHCII+/−. Concomitantly, after APP infection there was an increase in the PB MP CD14+ CD163+ CD203α− MHC II− population, suggesting that these cells give rise to inflammatory monocytes/macrophages. The CD203α and MHCII molecules appear on these cells after leaving the PB. In healthy animals, the BM MP precursors were represented by CD14− CD163− cells maturing directly into CD14+ CD163− that were then released into the PB. After infection, an altered maturation pathway of MP precursors appeared, represented by CD14− CD163− CD203α− MHCII− MP directly switching into CD14+ CD163+ CD203α− MHCII− MP. In conclusion, two different MP maturation pathways were suggested in pigs. The use of these pathways differs under inflammatory and noninflammatory conditions. PMID:20519113

  14. A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions.

    PubMed

    Savoye, C; Jobert, J L; Berthelot-Hérault, F; Keribin, A M; Cariolet, R; Morvan, H; Madec, F; Kobisch, M

    2000-05-11

    The study describes a polymerase chain reaction (PCR) assay for the detection of Actinobacillus pleuropneumoniae. The test is based on the amplification of the omlA gene coding for an outer membrane protein of A. pleuropneumoniae. To test the specificity of the reaction, 19 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR assay. The detection threshold of the test was 10(2) A. pleuropneumoniae CFU/assay. The test was then applied to the detection of A. pleuropneumoniae from tonsillar biopsies and tracheobronchial lavage fluids of pigs without a culture step. The detection of A. pleuropneumoniae in these samples was performed by PCR, by conventional culture and by bacteriology with immunomagnetic beads. The number of samples that were found positive by PCR was almost three times higher than the number of samples from which A. pleuropneumoniae was isolated by both bacteriological techniques. The detection of A. pleuropneumoniae in these samples allowed us to demonstrate its aerosol transmission to pigs under experimental conditions. The trial involved 18 specific pathogen free pigs. Six pigs, infected with A. pleuropneumoniae, were located in a unit A, together with four non-infected animals (contact pigs). Eight non-infected pigs (reporter pigs) were located in a unit B, adjacent to A. We detected A. pleuropneumoniae in samples from infected animals but also from 'contact' (unit A) and 'reporter' (unit B) pigs. The results of this study show that the simple preparation of the samples followed by the PCR assay may be a useful tool for epidemiological studies. PMID:10781732

  15. The Adh adhesin domain is required for trimeric autotransporter Apa1-mediated Actinobacillus pleuropneumoniae adhesion, autoaggregation, biofilm formation and pathogenicity.

    PubMed

    Wang, Lei; Qin, Wanhai; Yang, Shuxin; Zhai, Ruidong; Zhou, Liang; Sun, Changjiang; Pan, Fengguang; Ji, Qun; Wang, Yu; Gu, Jingmin; Feng, Xin; Du, Chongtao; Han, Wenyu; Langford, P R; Lei, Liancheng

    2015-05-15

    Actinobacillus pleuropneumoniae is a causative agent of porcine pleuropneumonia, which is a highly contagious endemic disease of pigs. Adhesion is a critical first step in the infection process. Trimeric autotransporter adhesions (TAAs) have been identified as novel virulence factors; however, little is known on their roles in A. pleuropneumoniae pathogenicity. Here, our data show that YadA-like head region (Adh) of Apa1 was the optimal adhesion functional domain via segment expression and adhesion assays in vitro. Additionally, Adh induced partial protection against A. pleuropneumoniae 5b L20 and serotypes 1, 3, and 5a in mice. The deletion of Adh gene significantly decreased autoaggregation, biofilm formation and adherence to host cells in vitro. Furthermore, with delaying of clinical symptoms, reducing production of pro-inflammatory cytokines and lessening the lung injury after infection, Adh deletion strain (5bϕAdh) significantly reduced the pathogenicity to piglets. To elucidate the mechanism of lung injury, the differentially expressed genes in the lung tissues of piglets infected with the 5b L20 or 5bϕAdh strains were investigated using microarray analysis and validated by qRT-PCR. Compared with the 5b L20 infected piglets, 495 genes were differentially expressed in 5bϕAdh infected lung tissue (221 upregulated and 274 downregulated). Especially, the antigen processing and presentation gene IFI30 was increased following infection with the 5bϕAdh strain. Thus, Adh may enhance pathogenicity by depressing host immune recognition. We conclude that the head domain of the A. pleuropneumoniae trimeric autotransporter Apa1 regulates autoagglutination, biofilm formation, adhesion to host cells and pathogenicity.

  16. Analysis of the Actinobacillus pleuropneumoniae ArcA regulon identifies fumarate reductase as a determinant of virulence.

    PubMed

    Buettner, Falk F R; Bendallah, Ibrahim M; Bosse, Janine T; Dreckmann, Karla; Nash, John H E; Langford, Paul R; Gerlach, Gerald-F

    2008-06-01

    The ability of the bacterial pathogen Actinobacillus pleuropneumoniae to grow anaerobically allows the bacterium to persist in the lung. The ArcAB two-component system is crucial for metabolic adaptation in response to anaerobic conditions, and we recently showed that an A. pleuropneumoniae arcA mutant had reduced virulence compared to the wild type (F. F. Buettner, A. Maas, and G.-F. Gerlach, Vet. Microbiol. 127:106-115, 2008). In order to understand the attenuated phenotype, we investigated the ArcA regulon of A. pleuropneumoniae by using a combination of transcriptome (microarray) and proteome (two-dimensional difference gel electrophoresis and subsequent mass spectrometry) analyses. We show that ArcA negatively regulates the expression of many genes, including those encoding enzymes which consume intermediates during fumarate synthesis. Simultaneously, the expression of glycerol-3-phosphate dehydrogenase, a component of the respiratory chain serving as a direct reduction equivalent for fumarate reductase, was upregulated. This result, together with the in silico analysis finding that A. pleuropneumoniae has no oxidative branch of the citric acid cycle, led to the hypothesis that fumarate reductase might be crucial for virulence by providing (i) energy via fumarate respiration and (ii) succinate and other essential metabolic intermediates via the reductive branch of the citric acid cycle. To test this hypothesis, an isogenic A. pleuropneumoniae fumarate reductase deletion mutant was constructed and studied by using a pig aerosol infection model. The mutant was shown to be significantly attenuated, thereby strongly supporting a crucial role for fumarate reductase in the pathogenesis of A. pleuropneumoniae infection.

  17. Transcriptional Portrait of Actinobacillus pleuropneumoniae during Acute Disease - Potential Strategies for Survival and Persistence in the Host

    PubMed Central

    Klitgaard, Kirstine; Friis, Carsten; Jensen, Tim K.; Angen, Øystein; Boye, Mette

    2012-01-01

    Background Gene expression profiles of bacteria in their natural hosts can provide novel insight into the host-pathogen interactions and molecular determinants of bacterial infections. In the present study, the transcriptional profile of the porcine lung pathogen Actinobacillus pleuropneumoniae was monitored during the acute phase of infection in its natural host. Methodology/Principal Findings Bacterial expression profiles of A. pleuropneumoniae isolated from lung lesions of 25 infected pigs were compared in samples taken 6, 12, 24 and 48 hours post experimental challenge. Within 6 hours, focal, fibrino hemorrhagic lesions could be observed in the pig lungs, indicating that A. pleuropneumoniae had managed to establish itself successfully in the host. We identified 237 differentially regulated genes likely to encode functions required by the bacteria for colonization and survival in the host. This group was dominated by genes involved in various aspects of energy metabolism, especially anaerobic respiration and carbohydrate metabolism. Remodeling of the bacterial envelope and modifications of posttranslational processing of proteins also appeared to be of importance during early infection. The results suggested that A. pleuropneumoniae is using various strategies to increase its fitness, such as applying Na+ pumps as an alternative way of gaining energy. Furthermore, the transcriptional data provided potential clues as to how A. pleuropneumoniae is able to circumvent host immune factors and survive within the hostile environment of host macrophages. This persistence within macrophages may be related to urease activity, mobilization of various stress responses and active evasion of the host defenses by cell surface sialylation. Conclusions/Significance The data presented here highlight the importance of metabolic adjustments to host conditions as virulence factors of infecting microorganisms and help to provide insight into the mechanisms behind the efficient

  18. A 24-kDa cloned zinc metalloprotease from Actinobacillus pleuropneumoniae is common to all serotypes and cleaves actin in vitro.

    PubMed Central

    García-Cuéllar, C; Montañez, C; Tenorio, V; Reyes-Esparza, J; Durán, M J; Negrete, E; Guerrero, A; de la Garza, M

    2000-01-01

    Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence that.codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 6. Figure 7. PMID:10805246

  19. Actinobacillus suis strains isolated from healthy and diseased swine are clonal and carry apxICABDvar. suis and apxIICAvar. suis toxin genes.

    PubMed Central

    Van Ostaaijen, J; Frey, J; Rosendal, S; MacInnes, J I

    1997-01-01

    Actinobacillus suis isolates recovered from both healthy and diseased pigs were characterized by biochemical testing, serotyping, restriction endonuclease fingerprinting, and apx toxin gene typing. The clinical isolates analyzed were collected over a 10-year period from approximately 40 different locations in southwestern Ontario, Canada. Little variation in the biochemical profiles of these isolates was seen, and all isolates reacted strongly with rabbit antisera prepared against one of the strains. Similarly, by using BamHI and BglII for restriction endonuclease fingerprinting (REF) analysis, all isolates were found to belong to a single REF group. Minor variations could be detected, especially in the BglII fingerprints, but overall the patterns were remarkably similar. Sequences that could be amplified by PCR with primers to the apxICA and apxIICA genes of Actinobacillus pleuropneumoniae were detected in all strains. Although no amplification was obtained with primers to the A. pleuropneumoniae apxIBD genes, sequences with homology to apxIBD were detected by hybridization. There was no evidence of apxIII homologs. Taken together, these data suggest that A. suis isolates are genotypically and phenotypically very similar, regardless of their source, and that they contain genes similar to, but not identical to, the apxICABD and apxIICA genes of A. pleuropneumoniae. PMID:9114394

  20. Characterization of the binding of Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) to glycosphingolipids, using a solid-phase overlay approach

    SciTech Connect

    Stroemberg, N.K.; Karlsson, K.A. )

    1990-07-05

    Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) were radiolabeled externally (125I) or metabolically (35S) and analyzed for their ability to bind glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells. Two binding properties were found and characterized in detail. (i) Both bacteria showed binding to lactosylceramide (LacCer) in a fashion similar to bacteria characterized earlier. The activity of free LacCer was dependent on the ceramide structure; species with 2-hydroxy fatty acid and/or a trihydroxy base were positive, while species with nonhydroxy fatty acid and a dihydroxy base were negative binders. Several glycolipids with internal lactose were active but only gangliotriaosylceramide and gangliotetraosylceramide were as active as free LacCer. The binding to these three species was half-maximal at about 200 ng of glycolipid and was not blocked by preincubation of bacteria with free lactose or lactose-bovine serum albumin. (ii) A. naeslundii, unlike A. viscosus, showed a superimposed binding concluded to be to terminal or internal GalNAc beta and equivalent to a lactose-inhibitable specificity previously analyzed by other workers. Terminal Gal beta was not recognized in several glycolipids, although free Gal and lactose were active as soluble inhibitors. The binding was half-maximal at about 10 ng of glycolipid. A glycolipid mixture prepared from a scraping of human buccal epithelium contained an active glycolipid with sites for both binding specificities.

  1. Efficacy of oral Bifidobacterium bifidum ATCC 29521 on microflora and antioxidant in mice.

    PubMed

    Wang, Bao-gui; Xu, Hai-bo; Xu, Feng; Zeng, Zhe-ling; Wei, Hua

    2016-03-01

    This study aimed to examine whether Bifidobacterium bifidum ATCC 29521, a species of colonic microflora in humans, is involved in the intestinal tract of mice. This study was also conducted to determine the antioxidant activity of this species by evaluating different microbial populations and reactive oxygen species isolated from feces and intestinal contents for 28 days of oral administration. Microbial diversities were assessed through bacterial culture techniques, PCR-DGGE, and real-time PCR. This study showed that the intake of B. bifidum ATCC 29521 significantly (p < 0.05) improved the ecosystem of the intestinal tract of BALB/c mice by increasing the amount of probiotics (Lactobacillus intestinalis and Lactobacillus crispatus) and by reducing unwanted bacterial populations (Enterobacter, Escherichia coli). Antioxidative activities of incubated cell-free extracts were evaluated through various assays, including the scavenging ability of DPPH radical (64.5% and 67.54% (p < 0.05), respectively, at 21 days in nutrients and 28 days in MRS broth), superoxide anion, and hydroxyl radical (85% and 61.5% (p < 0.05), respectively, at intestinal contents in nutrients and 21 days in MRS broth). Total reducing power (231.5 μmol/L (p < 0.05), 14 days in MRS broth) and mRNA level of genes related to oxidative stress were also determined. Results indicated that B. bifidum ATCC 29521 elicits a beneficial effect on murine gut microbiota and antioxidant activities compared with the control samples. This species can be considered as a potential bioresource antioxidant to promote health. Bifidobacterium bifidum ATCC 29521 may also be used as a promising material in microbiological and food applications. PMID:26863255

  2. Complete Genome Sequence of Pseudomonas syringae pv. lapsa Strain ATCC 10859, Isolated from Infected Wheat

    PubMed Central

    Kong, Jun; Jiang, Hongshan; Li, Baiyun; Zhao, Wenjun

    2016-01-01

    Pseudomonas syringae pv. lapsa is a pathovar of Pseudomonas syringae that can infect wheat. The complete genome of P. syringae pv. lapsa strain ATCC 10859 contains a 5,918,899-bp circular chromosome with 4,973 coding sequences, 16 rRNAs, 69 tRNAs, and an average GC content of 59.13%. The analysis of this genome revealed several gene clusters that are related to pathogenesis and virulence. PMID:26941133

  3. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. PMID:26168906

  4. Complete genome sequence of a malodorant-producing acetogen, Clostridium scatologenes ATCC 25775(T).

    PubMed

    Zhu, Zhengang; Guo, Ting; Zheng, Huajun; Song, Tianshun; Ouyang, Pingkai; Xie, Jingjing

    2015-10-20

    Clostridium scatologenes ATCC 25775(T) is an acetogenic anaerobic bacteria known to be capable of synthesizing volatile fatty acids and solvents from CO2 or CO on its autotrophic mode and producing 3-methylindole and 4-methylphenol on its heterotrophic mode. Here, we report the complete genome sequence of this strain, which might provide a lot of valuable information for developing metabolic engineering strategies to produce biofuels or chemicals from greenhouse gases. PMID:26210291

  5. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

  6. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Krovvidi, Ravi K.; Gritsenko, Marina A.; Monroe, Matthew E.; Moore, Ronald J.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2011-12-01

    Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis reveals fundamental insights into the control and regulation of these functions. To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Analysis of protein functions revealed that the expression of nitrogenase in the dark is mediated by higher respiration and glycogen metabolism. We have also shown that Cyanothece ATCC51142 utilizes alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. In conclusion, this study provides a deeper insight into how Cyanothece ATCC51142 modulates cellular functions to accommodate photosynthesis and N2-fixation within the single cell.

  7. Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Welsh, Eric A.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2012-02-03

    Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a 13C15N-L-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 422 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly and degradation showed higher levels of isotope incorporation suggesting that these biochemical pathways are important for growth under non-diazotrophic conditions. Calculation of relative isotope abundances (RIA) values allowed to measure actual active protein synthesis over time for different biochemical pathways under non-diazotrophic conditions. Overall results demonstrated the utility of 'non-steady state' pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

  8. Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579

    PubMed Central

    Abfalter, Carmen M.; Schönauer, Esther; Ponnuraj, Karthe; Huemer, Markus; Gadermaier, Gabriele; Regl, Christof; Briza, Peter; Ferreira, Fatima; Huber, Christian G.; Brandstetter, Hans; Posselt, Gernot; Wessler, Silja

    2016-01-01

    Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications. PMID:27588686

  9. Genome-scale reconstruction of metabolic networks of Lactobacillus casei ATCC 334 and 12A.

    PubMed

    Vinay-Lara, Elena; Hamilton, Joshua J; Stahl, Buffy; Broadbent, Jeff R; Reed, Jennifer L; Steele, James L

    2014-01-01

    Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications.

  10. Genome-scale reconstruction of metabolic networks of Lactobacillus casei ATCC 334 and 12A.

    PubMed

    Vinay-Lara, Elena; Hamilton, Joshua J; Stahl, Buffy; Broadbent, Jeff R; Reed, Jennifer L; Steele, James L

    2014-01-01

    Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications. PMID:25365062

  11. Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

    PubMed Central

    Gaballa, A; Abeysinghe, P D; Urich, G; Matthijs, S; De Greve, H; Cornelis, P; Koedam, N

    1997-01-01

    Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism. PMID:9361421

  12. Degradation of nitrocellulose-based paint by Desulfovibrio desulfuricans ATCC 13541.

    PubMed

    Giacomucci, L; Toja, F; Sanmartín, P; Toniolo, L; Prieto, B; Villa, F; Cappitelli, F

    2012-09-01

    Nitrocellulose is one of the most commonly used compounds in ammunition and paint industries and its recalcitrance to degradation has a negative impact on human health and the environment. In this study the capability of Desulfovibrio desulfuricans ATCC 13541 to degrade nitrocellulose as binder in paint was assayed for the first time. Nitrocellulose-based paint degradation was followed by monitoring the variation in nitrate, nitrite and ammonium content in the culture medium using Ultraviolet-Visible spectroscopy. At the same time cell counts and ATP assay were performed to estimate bacterial density and activity in all samples. Infrared spectroscopy and colorimetric measurements of paint samples were performed to assess chemical and colour changes due to the microbial action. Microscope observations of nitrocellulose-based paint samples demonstrated the capability of the bacterium to adhere to the paint surface and change the paint adhesive characteristics. Finally, preliminary studies of nitrocellulose degradation pathway were conducted by assaying nitrate- and nitrite reductases activity in D. desulfuricans grown in presence or in absence of paint. We found that D. desulfuricans ATCC 13541 is able to transform nitrocellulose as paint binder and we hypothesised ammonification as degradation pathway. The results suggest that D. desulfuricans ATCC 13541 is a good candidate as a nitrocellulose-degrading bacterium.

  13. Genome –Scale Reconstruction of Metabolic Networks of Lactobacillus casei ATCC 334 and 12A

    PubMed Central

    Vinay-Lara, Elena; Hamilton, Joshua J.; Stahl, Buffy; Broadbent, Jeff R.; Reed, Jennifer L.; Steele, James L.

    2014-01-01

    Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications. PMID:25365062

  14. Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T

    PubMed Central

    Iiyama, Kazuhiro; Mon, Hiroaki; Mori, Kazuki; Mitsudome, Takumi; Lee, Jae Man; Kusakabe, Takahiro; Tashiro, Kousuke; Asano, Shin-ichiro; Yasunaga-Aoki, Chisa

    2015-01-01

    A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706T shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706T were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706T and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage. PMID:25853059

  15. Characterization of enzymatic reduction of hexavalent chromium by Escherichia coli ATCC 33456.

    PubMed Central

    Shen, H; Wang, Y T

    1993-01-01

    Chromium reduction by Escherichia coli ATCC 33456 quantitatively transferred hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III). The reduced chromium was predominantly present in the external medium. Supernatant fluids of cell extract, obtained by centrifugation at 12,000 and 150,000 x g, showed almost the same Cr(VI) reduction activity, indicating that Cr(VI) reduction by E. coli ATCC 33456 was a largely soluble reductase activity. In studies with respiratory inhibitors, no inhibitory effects on aerobic and anaerobic Cr(VI) reduction were demonstrated by addition of cyanide, azide, and rotenone into both intact cell cultures and supernatant fluids of E. coli ATCC 33456. Although cytochromes b and d were identified in the membrane fraction of cell extracts, Cr(VI) was not reduced by the membrane fraction alone. The cytochrome difference spectra analysis also indicated that these cytochromes of the respiratory chain require the presence of the soluble Cr(VI) reductase to mediate electron transport to Cr(VI). Stimulation of Cr(VI) reduction by an uncoupler, 2,4-dinitrophenol, indicated that the respiratory-chain-linked electron transport to Cr(VI) was limited by the rate of dissipation of the proton motive force. PMID:8285683

  16. Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579.

    PubMed

    Abfalter, Carmen M; Schönauer, Esther; Ponnuraj, Karthe; Huemer, Markus; Gadermaier, Gabriele; Regl, Christof; Briza, Peter; Ferreira, Fatima; Huber, Christian G; Brandstetter, Hans; Posselt, Gernot; Wessler, Silja

    2016-01-01

    Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications. PMID:27588686

  17. Actinobacillus pleuropneumoniae genes expression in biofilms cultured under static conditions and in a drip-flow apparatus

    PubMed Central

    2013-01-01

    Background Actinobacillus pleuropneumoniae is the Gram-negative bacterium responsible for porcine pleuropneumonia. This respiratory infection is highly contagious and characterized by high morbidity and mortality. The objectives of our study were to study the transcriptome of A. pleuropneumoniae biofilms at different stages and to develop a protocol to grow an A. pleuropneumoniae biofilm in a drip-flow apparatus. This biofilm reactor is a system with an air-liquid interface modeling lung-like environment. Bacteria attached to a surface (biofilm) and free floating bacteria (plankton) were harvested for RNA isolation. Labelled cDNA was hybridized to a microarray to compare the expression profiles of planktonic cells and biofilm cells. Results It was observed that 47 genes were differentially expressed (22 up, 25 down) in a 4 h-static growing/maturing biofilm and 117 genes were differentially expressed (49 up, 68 down) in a 6h-static dispersing biofilm. The transcriptomes of a 4 h biofilm and a 6 h biofilm were also compared and 456 genes (235 up, 221 down) were identified as differently expressed. Among the genes identified in the 4 h vs 6h biofilm experiment, several regulators of stress response were down-regulated and energy metabolism associated genes were up-regulated. Biofilm bacteria cultured using the drip-flow apparatus differentially expressed 161 genes (68 up, 93 down) compared to the effluent bacteria. Cross-referencing of differentially transcribed genes in the different assays revealed that drip-flow biofilms shared few differentially expressed genes with static biofilms (4 h or 6 h) but shared several differentially expressed genes with natural or experimental infections in pigs. Conclusion The formation of a static biofilm by A. pleuropneumoniae strain S4074 is a rapid process and transcriptional analysis indicated that dispersal observed at 6 h is driven by nutritional stresses. Furthermore, A. pleuropneumoniae can form a biofilm under low

  18. Immunological study of an attenuated Salmonella Typhimurium expressing ApxIA, ApxIIA, ApxIIIA and OmpA of Actinobacillus pleuropneumoniae in a mouse model.

    PubMed

    Hur, Jin; Eo, Seong Kug; Park, Sang-Youel; Choi, Yoonyoung; Lee, John Hwa

    2016-01-01

    Salmonella Typhimurium strain expressing the Actinobacillus pleuropneumoniae antigens, ApxIA, ApxIIA, ApxIIIA and OmpA, was previously constructed as a vaccine candidate for porcine pleuropneumonia. This strain was a live attenuated (∆lon∆cpxR∆asd)Salmonella as a delivery host and contained a vector containing asd. An immunological study of lymphocyte proliferation, T-lymphocyte subsets and cytokines in the splenocytes of a mouse model was carried out after stimulation with the candidate Salmonella Typhimurium by intranasal inoculation. The splenic lymphocyte proliferation and the levels of IL-4, IL-6 and IL-12 of the inoculated mice were significantly increased, and the T- and B-cell populations were also elevated. Collectively, the candidate may efficiently induce the Th1- and Th2-type immune responses.

  19. Minimum inhibitory concentration breakpoints and disk diffusion inhibitory zone interpretive criteria for tilmicosin susceptibility testing against Pasteurella multocida and Actinobacillus pleuropneumoniae associated with porcine respiratory disease.

    PubMed

    Shryock, Thomas R; Staples, J Mitchell; DeRosa, David C

    2002-09-01

    Tilmicosin is a novel macrolide antibiotic developed for exclusive use in veterinary medicine. Tilmicosin has been approved as a feed premix to control porcine respiratory disease associated with Pasteurella multocida and Actinobacillus pleuropneumoniae. The development of antimicrobial susceptibility testing guidelines for tilmicosin was predicated on the relationship of clinical efficacy studies that demonstrated a favorable therapeutic outcome, on pharmacokinetic data, and on in vitro test data, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The approved breakpoints for the minimum inhibitory concentration dilution testing for both species are resistant, > or = 32 microg/ml, and susceptible, < or = 16 microg/ml. The zone of inhibition interpretive criteria for disk diffusion testing with a 15-microg tilmicosin disk are resistant, < or = 10 mm, and susceptible, > or = 11 mm.

  20. Investigation of the Amycolatopsis sp. strain ATCC 39116 vanillin dehydrogenase and its impact on the biotechnical production of vanillin.

    PubMed

    Fleige, Christian; Hansen, Gunda; Kroll, Jens; Steinbüchel, Alexander

    2013-01-01

    The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDH(ATCC 39116)). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDH(ATCC 39116) was purified to apparent electrophoretic homogeneity and exhibited NAD(+)-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin.

  1. Investigation of the Amycolatopsis sp. strain ATCC 39116 vanillin dehydrogenase and its impact on the biotechnical production of vanillin.

    PubMed

    Fleige, Christian; Hansen, Gunda; Kroll, Jens; Steinbüchel, Alexander

    2013-01-01

    The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDH(ATCC 39116)). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDH(ATCC 39116) was purified to apparent electrophoretic homogeneity and exhibited NAD(+)-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin. PMID:23064333

  2. Improved penicillin amidase production using a genetically engineered mutant of escherichia coli ATCC 11105

    SciTech Connect

    Robas, N.; Zouheiry, H.; Branlant, G.; Branlant, C. )

    1993-01-05

    Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, the authors constructed various recombinant E. coli HB 101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic acid (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the HindIII fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.

  3. Evaluating Chemical Mitigation of Salmonella Typhimurium ATCC 14028 in Animal Feed Ingredients.

    PubMed

    Cochrane, Roger A; Huss, Anne R; Aldrich, Gregory C; Stark, Charles R; Jones, Cassandra K

    2016-04-01

    Salmonella Typhimurium is a potential feed safety hazard in animal feed ingredients. Thermal mitigation of Salmonella spp. during rendering is effective but does not eliminate the potential for cross-contamination. Therefore, the objective of this experiment was to evaluate the effectiveness of chemicals to mitigate postrendering Salmonella Typhimurium ATCC 14028 contamination in rendered proteins over time. Treatments were arranged in a 6 × 4 factorial with six chemical treatments and four rendered protein meals. The chemical treatments included (i) control without chemical treatment, (ii) 0.3% commercial formaldehyde product, (iii) 2% essential oil blend, (iv) 2% medium chain fatty acid blend, (v) 3% organic acid blend, and (vi) 1% sodium bisulfate. The four rendered protein meals included (i) feather meal, (ii) blood meal, (iii) meat and bone meal, and (iv) poultry by-product meal. After matrices were chemically treated, they were inoculated with Salmonella Typhimurium ATCC 14028, stored at room temperature, and enumerated via plate counts on days 0, 1, 3, 7, 14, 21, and 42 postinoculation. The Salmonella concentration in ingredients treated with medium chain fatty acid and commercial formaldehyde were similar to one another (P = 0.23) but were 2 log lower than the control (P < 0.05). Ingredients treated with organic acids and essential oils also had lower Salmonella concentrations than the control (P < 0.05). Time also played a significant role in Salmonella mitigation, because all days except days 14 and 21 (P = 0.92) differed from one another. Rendered protein matrix also affected Salmonella stability, because concentrations in meat and bone meal and blood meal were similar to one another (P = 0.36) but were greater than levels in feather meal and poultry by-product meal (P < 0.05). In summary, chemical treatment and time both mitigated Salmonella Typhimurium ATCC 14028, but their effectiveness was matrix dependent. Time and chemical treatment with medium

  4. Multicenter Investigation of Gepotidacin (GSK2140944) Agar Dilution Quality Control Determinations for Neisseria gonorrhoeae ATCC 49226.

    PubMed

    Jones, Ronald N; Fedler, Kelley A; Scangarella-Oman, Nicole E; Ross, James E; Flamm, Robert K

    2016-07-01

    Gepotidacin, a novel triazaacenaphthylene antibacterial agent, is the first in a new class of type IIA topoisomerase inhibitors with activity against many biothreat and conventional pathogens, including Neisseria gonorrhoeae To assist ongoing clinical studies of gepotidacin to treat gonorrhea, a multilaboratory quality assurance investigation determined the reference organism (N. gonorrhoeae ATCC 49226) quality control MIC range to be 0.25 to 1 μg/ml (88.8% of gepotidacin MIC results at the 0.5 μg/ml mode). PMID:27161642

  5. Complete genome sequence of Vibrio alginolyticus ATCC 33787(T) isolated from seawater with three native megaplasmids.

    PubMed

    Wang, Pengxia; Wen, Zhongling; Li, Baiyuan; Zeng, Zhenshun; Wang, Xiaoxue

    2016-08-01

    Vibrio alginolyticus, an opportunistic pathogen, is commonly associated with vibriosis in fish and shellfish and can also cause superficial and ear infections in humans. V. alginolyticus ATCC 33787(T) was originally isolated from seawater and has been used as one of the type strains for exploring the virulence factors of marine bacteria and for developing vaccine against vibriosis. Here we sequenced and assembled the whole genome of this strain, and identified three megaplasmids and three Type VI secretion systems, thus providing useful information for the study of virulence factors and for the development of vaccine for Vibrio.

  6. Production of fructosyltransferase by Aureobasidium sp. ATCC 20524 in batch and two-step batch cultures.

    PubMed

    Salinas, Martín A; Perotti, Nora I

    2009-01-01

    A comparison of fructosyltransferase (EC 2.4.1.9) production by Aureobasidium sp. ATCC 20524 in batch and two step batch cultures was investigated in a 1-l stirred tank reactor using a sucrose supply of 200 g/l. Results showed that the innovative cultivation in two step of Aureobasidium sp. produced more fructosyltransferase (FFase) than the single batch culture at the same sucrose concentration with a maximal enzyme production of 523 U/ml, which was 80.5% higher than the one obtained in the batch culture. The production of fructooligosaccharides (FOSs) was also analyzed; their concentration reached a maximum value of 160 g/l the first day in the two-step culture and 127 g/l in the single-batch mode. The use of the two-step batch culture with Aureobasidium sp. ATCC 20524 in allowing the microorganism to grow up prior to the induction of sucrose (second step), proved to be a powerful method for producing fructosyltransferase and FOSs. PMID:18810518

  7. An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, D; Mayer, D A; Moritz, D E; Oliveira, D; de Souza, A A Ulson; Souza, S M A Guelli

    2015-10-01

    Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process. PMID:26201481

  8. Cloning, expression, and sequencing of a protease gene from Bacteroides forsythus ATCC 43037 in Escherichia coli.

    PubMed Central

    Saito, T; Ishihara, K; Kato, T; Okuda, K

    1997-01-01

    We have isolated and characterized an N-benzoyl-Val-Gly-Arg-p-nitroanilide-specific protease gene, designated prtH, from Bacteroides forsythus ATCC 43037. Nucleotide sequencing of the DNA insert from the clone (hereafter referred to as clone FST) revealed that the protease activity corresponded to an open reading frame consisting of 1,272 bp coding for a 47.8-kDa protein. When plasmid pFST was used as a probe in Southern hybridization, Sau3AI-digested chromosomal DNA of B. forsythus ATCC 43037 as well as the chromosomal DNAs of the isolated strains Ta4, TR5, and YG2 showed 0.6- and 0.8-kb hybridizing bands. The cell-free extracts of clone FST showed hemolytic activity on human blood cells. The hydrolytic activity of cell extracts of the pFST clone was inhibited by p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, N-ethylmaleimide, iodoacetic acid, iodoaceteamide, and EDTA. PMID:9353083

  9. Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

    PubMed

    Pérez-Rodríguez, Noelia; Oliveira, Ricardo Pinheiro de Souza; Agrasar, Ana María Torrado; Domínguez, José Manuel

    2016-02-01

    The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds. PMID:26476645

  10. Genome Sequence of Aeromonas hydrophila ATCC 7966T: Jack of All Trades▿

    PubMed Central

    Seshadri, Rekha; Joseph, Sam W.; Chopra, Ashok K.; Sha, Jian; Shaw, Jonathan; Graf, Joerg; Haft, Daniel; Wu, Martin; Ren, Qinghu; Rosovitz, M. J.; Madupu, Ramana; Tallon, Luke; Kim, Mary; Jin, Shaohua; Vuong, Hue; Stine, O. Colin; Ali, Afsar; Horneman, Amy J.; Heidelberg, John F.

    2006-01-01

    The complete genome of Aeromonas hydrophila ATCC 7966T was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966T. Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments. PMID:16980456

  11. In silico analysis of Clostridium acetobutylicum ATCC 824 metabolic response to an external electron supply.

    PubMed

    Gallardo, Roberto; Acevedo, Alejandro; Quintero, Julián; Paredes, Ivan; Conejeros, Raúl; Aroca, Germán

    2016-02-01

    The biological production of butanol has become an important research field and thanks to genome sequencing and annotation; genome-scale metabolic reconstructions have been developed for several Clostridium species. This work makes use of the iCAC490 model of Clostridium acetobutylicum ATCC 824 to analyze its metabolic capabilities and response to an external electron supply through a constraint-based approach using the Constraint-Based Reconstruction Analysis Toolbox. Several analyses were conducted, which included sensitivity, production envelope, and phenotypic phase planes. The model showed that the use of an external electron supply, which acts as co-reducing agent along with glucose-derived reducing power (electrofermentation), results in an increase in the butanol-specific productivity. However, a proportional increase in the butyrate uptake flux is required. Besides, the uptake of external butyrate leads to the coupling of butanol production and growth, which coincides with results reported in literature. Phenotypic phase planes showed that the reducing capacity becomes more limiting for growth at high butyrate uptake fluxes. An electron uptake flux allows the metabolism to reach the growth optimality line. Although the maximum butanol flux does not coincide with the growth optimality line, a butyrate uptake combined with an electron uptake flux would result in an increased butanol volumetric productivity, being a potential strategy to optimize the production of butanol by C. acetobutylicum ATCC 824.

  12. Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

    PubMed

    Pérez-Rodríguez, Noelia; Oliveira, Ricardo Pinheiro de Souza; Agrasar, Ana María Torrado; Domínguez, José Manuel

    2016-02-01

    The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds.

  13. Media optimization studies for Serratiopeptidase production from Serratia marcescens ATCC 13880.

    PubMed

    Badhe, Ravindra V; Nanda, Rabindra K; Kulkarni, Manasi B; Bhujbal, Mayur N; Patil, Pradeep S; Badhe, Sonali R

    2009-01-01

    Production of an anti-inflammatory enzyme serratiopeptidase by fermentation with Serratia marcescens ATCC 13880 was studied to ascertain optimal nutritional conditions for large scale production. To study biosynthesis and production of serratiopeptidase by Serratia marcescens ATCC 13880, different physicochemical parameters were studied and optimized. The optimized medium contain, (g/l) glycerine 10.0, maltose 10.0 as carbon source, peptone 10.0 as organic nitrogen source, ammonium sulphate 10.0 as inorganic nitrogen source, dihydrogen phosphate 10.0, sodium bicarbonate 10.0, sodium acetate 10.0 as inorganic salt source, ascorbic acid 10.0 as stabilizer, distilled water 1000 ml and the optimized fermentation conditions were pH 7.0, temperature 37 degrees C and duration 24 hr. The modified fermentation medium produced 27.36 IU/ml of serratiopeptidase compared to 17.97 IU/ml in basal medium and the molecular weight of the purified serratiopeptidase was found to be 52 kD.

  14. The Glycopeptide Antitumor Antibiotic Zorbamycin from Streptomyces flavoviridis ATCC 21892: Strain Improvement and Structure Elucidation⊥

    PubMed Central

    Wang, Liyan; Yun, Bong-Sik; George, Nicholas P.; Wendt-Pienkowski, Evelyn; Galm, Ute; Oh, Tae-Jin; Coughlin, Jane M.; Zhang, Guodong; Tao, Meifeng; Shen, Ben

    2008-01-01

    Zorbamycin (1, ZBM) is a glycopeptide antitumor antibiotic first reported in 1971. The partial structures of 1 were speculated on the basis of its acid hydrolysis products, but the structure of the intact molecule has never been established. The low titer of 1 from the wild-type strains, combined with its acid-instability, has so far hampered its isolation. By random mutagensis of Streptomyces flavoviridis ATCC21892, a wild-type producer of 1, with UV irradiation, two high-producing strains of 1, S. flavoviridis SB9000 and SB9001, were isolated. Under the optimized fermentation conditions, these two strains produced about 10 mg/L of 1, which was about 10-fold higher than the wild-type ATCC21892 strain, as estimated by HPLC analysis. Finally, 1 was isolated both as a 1-Cu complex and Cu-free molecule, and the intact structure of 1 was established on the basis of a combination of mass spectrometry and 1H and 13C NMR spectroscopic analyses. PMID:17311457

  15. Assessment of in vitro removal of cholesterol oxidation products by Lactobacillus casei ATCC334.

    PubMed

    Machorro-Méndez, I A; Hernández-Mendoza, A; Cardenia, V; Rodriguez-Estrada, M T; Lercker, G; Spinelli, F; Cellini, A; García, H S

    2013-11-01

    Cholesterol oxidation products (COPs) are a group of compounds formed during processing and storage of foods from animal origin. After ingestion, COPs are absorbed in the intestine and can be distributed to serum and various tissues, potentially promoting a variety of toxic effects. Therefore, inhibition of their intestinal absorption may contribute to reduce the health risks associated with dietary intake of COPs. Some studies have shown that drugs and dietary compounds may inhibit the intestinal absorption of dietary COPs. However, proven cholesterol- and/or food toxins-binding lactic acid bacteria have not been previously evaluated as potential COPs removal agents. The aim of this study was to assess the ability of Lactobacillus casei ATCC334 to remove COPs in aqueous solution. Results showed the ability of both growing and resting cells to remove COPs (ca. 30-60%). All COPs-bacterium interactions were specific and partly reversible, being resting cells the most efficient for COPs removal in a ranking order of 7-KC > 7α-OH/7β-OH > triol > 5,6β-EP > 5,6α-EP > 25-OH. Binding to the cell wall and/or cell membrane incorporation appears to be the most likely mechanisms involved on COPs removal by L. casei ATCC 334.

  16. An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, D; Mayer, D A; Moritz, D E; Oliveira, D; de Souza, A A Ulson; Souza, S M A Guelli

    2015-10-01

    Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process.

  17. Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

    NASA Technical Reports Server (NTRS)

    Schneegurt, M. A.; Sherman, D. M.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

    1997-01-01

    Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.

  18. Isolation and Characterization of a Novel Virulent Phage of Lactobacillus casei ATCC 393.

    PubMed

    Zhang, Xi; Lan, Yu; Jiao, Wenchao; Li, Yijing; Tang, Lijie; Jiang, Yanping; Cui, Wen; Qiao, Xinyuan

    2015-12-01

    A new virulent phage (Lcb) of Lactobacillus casei ATCC 393 was isolated from Chinese sauerkraut. It was specific to L. casei ATCC 393. Electron micrograph revealed that it had an icosahedral head (60.2 ± 0.8 nm in diameter) and a long tail (251 ± 2.6 nm). It belonged to the Siphoviridae family. The genome of phage Lcb was estimated to be approximately 40 kb and did not contain cohesive ends. One-step growth kinetics of its lytic development revealed latent and burst periods of 75 and 45 min, respectively, with a burst size of 16 PFU per infected cell. The phage was able to survive in a pH range between 4 and 11. However, a treatment of 70 °C for 30 min and 75% ethanol or isopropanol for 20 min was observed to inactivate phage Lcb thoroughly. The presence of both Ca(2+) and Mg(2+) showed a little influence on phage adsorption, but they were indispensable to gain complete lysis and improve plaque formation. The adsorption kinetics were similar on viable or nonviable cells, and high adsorption rates maintained between 10 and 37 °C. The highest adsorption rate was at 30 °C. This study increased the knowledge on phages of L. casei. The characterization of phage Lcb is helpful to establish a basis for adopting effective strategies to control phage attack in industry. PMID:26123178

  19. Response of electrically stimulated cells of Pseudomonas oleovorans strain ATCC 29347 suspended in silicone oil.

    PubMed

    Anglade, J; Hirschler, A; Le Petit, J; Matheron, R; Scarpitta, A; Iacazio, G

    2001-05-15

    A high intensity direct current was applied for more than 10 min onto a bacterial suspension of Pseudomonas oleovorans ATCC 29347 suspended in silicone oil. The application of a gradually increased electric field from 0 to 2500 V x cm(-1) resulted in a decrease of the optical density of the bacterial suspension and the occurrence of a peak current of several hundred microA for living cells instead of a linear increase (few microA) for killed or lyophilised cells. This procedure is not only a rapid way of investigating the living state of cell cultures but also an efficient experimental tool to study the cellular effects of a controlled electrical stress. PMID:11356578

  20. Direct observation of redox reactions in Candida parapsilosis ATCC 7330 by Confocal microscopic studies

    PubMed Central

    Venkataraman, Sowmyalakshmi; Narayan, Shoba; Chadha, Anju

    2016-01-01

    Confocal microscopic studies with the resting cells of yeast, Candida parapsilosis ATCC 7330, a reportedly versatile biocatalyst for redox enzyme mediated preparation of optically pure secondary alcohols in high optical purities [enantiomeric excess (ee) up to >99%] and yields, revealed that the yeast cells had large vacuoles under the experimental conditions studied where the redox reaction takes place. A novel fluorescence method was developed using 1-(6-methoxynaphthalen-2-yl)ethanol to track the site of biotransformation within the cells. This alcohol, itself non-fluorescent, gets oxidized to produce a fluorescent ketone, 1-(6-methoxynaphthalen-2-yl)ethanone. Kinetic studies showed that the reaction occurs spontaneously and the products get released out of the cells in less time [5 mins]. The biotransformation was validated using HPLC. PMID:27739423

  1. Closing the Carbon Balance for Fermentation by Clostridium thermocellum (ATCC 27405)

    SciTech Connect

    Ellis, Lucas D; Holwerda, Evert K; Hogsett, David; Rogers, Steve; Shao, Xiongjun; Tschaplinski, Timothy J; Thorne, Phil; Lynd, L.

    2012-01-01

    Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, {>=}92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products - ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.

  2. Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Egli, C; Hancock, R E; Holt, S C

    1992-01-01

    Three major outer membrane proteins with apparent molecular masses of 43, 45, and 51 kDa were purified from Wolinella recta ATCC 33238, and their pore-forming abilities were determined by the black lipid bilayer method. The non-heat-modifiable 45-kDa protein (Omp 45) showed no pore-forming activity even at high KCl concentrations. The single-channel conductances in 1 M KCl of the heat-modifiable proteins with apparent molecular masses of 43 kDa (Omp 43) and 51 kDa (Omp 51) were 0.49 and 0.60 nS, respectively. The proteins formed nonselective channels and, as determined by experiments of ion selectivity and zero-current potential, were weakly anion selective. Images PMID:1370429

  3. L-serine enhances the anaerobic lactate metabolism of Veillonella dispar ATCC 17745.

    PubMed

    Hoshino, E

    1987-06-01

    Under anaerobic conditions, the rate of metabolism of lactate by starved resting cells of Veillonella dispar ATCC 17745 was very low. Because pyruvate was metabolized well by the starved cells, oxidation of lactate to pyruvate, which is the first step of the lactate metabolism, must have been limited in the cells. In the starved cells, the levels of the metabolic intermediates, oxalacetate or fumarate, of which reductions to malate or to succinate could be coupled with lactate oxidation to pyruvate and initiate lactate metabolism, were quite low, suggesting that these had been reduced during the starvation steps under strictly anaerobic conditions. Thus, the starved cells were unable to start the anaerobic lactate metabolism because of shortage of such reducible substrates. L-serine greatly enhanced anaerobic lactate metabolism of the starved cells. This enhancement may have been due to metabolism of L-serine itself and conversion to oxalacetate and fumarate, which made it possible to begin lactate oxidation.

  4. Ala(0)-actagardine, a new lantibiotic from cultures of Actinoplanes liguriae ATCC 31048.

    PubMed

    Vértesy, L; Aretz, W; Bonnefoy, A; Ehlers, E; Kurz, M; Markus, A; Schiell, M; Vogel, M; Wink, J; Kogler, H

    1999-08-01

    The actagardine-producing strain Actinoplanes liguriae ATCC 31048, forms an additional lantibiotic when it is cultured on mannitol and soya meal. The new compound, Ala(0)-actagardine (1), has been isolated by solid-phase extraction followed by a two-step chromatographic separation. The molecular formula of 1 is C84H129N21O25S4. Its chemical structure was determined by 2D-NMR analysis and was further confirmed by an amino acid analysis, Edman degradation, and partial synthesis from actagardine. 1 exhibits a slightly higher biological activity than the parent compound actagardine. The synthetic analogs Lys(0)-actagardine (2) and Ile(0)-actagardine (3) demonstrate also antibacterial activities and emphasize the importance of the N-terminus for further derivatization. PMID:10580386

  5. Microbial conversion of ethylbenzene to 1-phenethanol and acetophenone by Nocardia tartaricans ATCC 31190.

    PubMed

    Cox, D P; Goldsmith, C D

    1979-09-01

    A culture of Nocardia tartaricans ATCC 31190 was capable of catalyzing the conversion of ethylbenzene to 1-phenethanol and acetophenone while growing in a shake flask culture with hexadecane as the source of carbon and energy. This subterminal oxidative reaction with ethylbenzene appears not to have been previously reported for Nocardia species. When N. tartaricans was grown on glucose as its source of carbon and energy and ethylbenzene was added, no subsequent production of 1-phenethanol or acetophenone was observed. The mechanisms of 1-phenethanol and acetophenone production from ethylbenzene are thought to involve a subterminal oxidation of the alpha-carbon of the alkyl group to 1-phenethanol followed by biological oxidation of the latter to acetophenone.

  6. Pullulan Production by Aureobasidium pullulans ATCC 201253 Cells Adsorbed onto Cellulose Anion and Cation Exchangers

    PubMed Central

    West, Thomas P.

    2012-01-01

    The anion exchanger phosphocellulose and the cation exchanger triethylaminoethyl cellulose were used to immobilize cells of the fungus Aureobasidium pullulans ATCC 201253 and the adsorbed cells were subsequently investigated for their ability to produce the polysaccharide pullulan using batch fermentation. The cells adsorbed on the triethylaminoethyl cellulose at pH 7.5 produced higher pullulan levels than those cells immobilized on phosphocellulose at pH 4.0 for 2 cycles of 168 h at 30 °C. Relative to the initial cycle of 168 h, pullulan production by the cells immobilized on the triethylaminoethyl cellulose decreased slightly after 168 h of the second production cycle while pullulan production by the phosphocellulose-immobilized cells remained about the same after 168 h of the second production cycle. PMID:23762749

  7. Effects of Salt Stress on Carbohydrate Metabolism of Lactobacillus plantarum ATCC 14917.

    PubMed

    Wang, Pingping; Wu, Zhen; Wu, Jing; Pan, Daodong; Zeng, Xiaoqun; Cheng, Kemeng

    2016-10-01

    Lactic acid bacteria are widely used in fermented foods, especially cheese products. In this study, we observed the salt tolerance of Lactobacillus plantarum ATCC 14917 after exposure to different concentrations of NaCl in MRS medium. Quantitative proteomic profiles using two-dimensional electrophoresis identified 384 proteins, of which 26 were upregulated and 31 downregulated. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry was then used to identify 11 proteins, of which three were linked to carbohydrate metabolism. The downregulation of carbamoyl phosphate synthase in carbohydrate metabolism revealed a bacterial regulation mechanism to save energy in order to survive during the salt tolerance. Other proteins were found involved in transcription-translation processes, fatty acid biosynthesis, and the primary metabolic process.

  8. Biotransformation of the Antimelanoma Agent Betulinic Acid by Bacillus megaterium ATCC 13368

    PubMed Central

    Chatterjee, Parnali; Kouzi, Samir A.; Pezzuto, John M.; Hamann, Mark T.

    2000-01-01

    Microbial transformation of the antimelanoma agent betulinic acid was studied. The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound. Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11α-hydroxy-lup-20(29)-en-28-oic acid, 1β-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3β,7β,15α-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses. In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid). PMID:10966400

  9. Resistance to vanadium in Pseudomonas fluorescens ATCC 17400 caused by mutations in TCA cycle enzymes.

    PubMed

    Denayer, Sarah; Matthijs, Sandra; Cornelis, Pierre

    2006-11-01

    Vanadium inhibits the growth of Pseudomonas fluorescens ATCC 17400 in the low-iron casamino acids medium and even more when iron is added to the medium. Analysis of transposon mutants allowed the isolation of two mutants with increased resistance to vanadium. One mutant had an insertion in the idh gene coding for the tricarboxylic acid enzyme isocitrate dehydrogenase. The second mutant had the transposon inserted into acnD, one out of three genes coding for a 2-methyl-isocitrate dehydratase (aconitase). In this mutant, there was a higher level of acnB aconitase transcripts while the levels of acnA transcripts were unchanged. A nonpolar idh mutant was obtained, which showed the same level of resistance against vanadium as the original transposon mutant. PMID:17020548

  10. Effects of Salt Stress on Carbohydrate Metabolism of Lactobacillus plantarum ATCC 14917.

    PubMed

    Wang, Pingping; Wu, Zhen; Wu, Jing; Pan, Daodong; Zeng, Xiaoqun; Cheng, Kemeng

    2016-10-01

    Lactic acid bacteria are widely used in fermented foods, especially cheese products. In this study, we observed the salt tolerance of Lactobacillus plantarum ATCC 14917 after exposure to different concentrations of NaCl in MRS medium. Quantitative proteomic profiles using two-dimensional electrophoresis identified 384 proteins, of which 26 were upregulated and 31 downregulated. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry was then used to identify 11 proteins, of which three were linked to carbohydrate metabolism. The downregulation of carbamoyl phosphate synthase in carbohydrate metabolism revealed a bacterial regulation mechanism to save energy in order to survive during the salt tolerance. Other proteins were found involved in transcription-translation processes, fatty acid biosynthesis, and the primary metabolic process. PMID:27342422

  11. A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163.

    PubMed

    Mohedano, María de la Luz; Russo, Pasquale; de Los Ríos, Vivian; Capozzi, Vittorio; Fernández de Palencia, Pilar; Spano, Giuseppe; López, Paloma

    2014-02-26

    Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

  12. Complete genome sequence of Streptomyces venezuelae ATCC 15439, a promising cell factory for production of secondary metabolites.

    PubMed

    Song, Ju Yeon; Yoo, Young Ji; Lim, Si-Kyu; Cha, Sun Ho; Kim, Ji-Eun; Roe, Jung-Hye; Kim, Jihyun F; Yoon, Yeo Joon

    2016-02-10

    Streptomyces venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds. PMID:26718561

  13. Complete genome sequence of Streptomyces venezuelae ATCC 15439, a promising cell factory for production of secondary metabolites.

    PubMed

    Song, Ju Yeon; Yoo, Young Ji; Lim, Si-Kyu; Cha, Sun Ho; Kim, Ji-Eun; Roe, Jung-Hye; Kim, Jihyun F; Yoon, Yeo Joon

    2016-02-10

    Streptomyces venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds.

  14. Genome Sequence of Streptococcus phocae subsp. phocae Strain ATCC 51973T Isolated from a Harbor Seal (Phoca vitulina)

    PubMed Central

    Poblete-Morales, Matías

    2015-01-01

    Streptococcus phocae subsp. phocae is a pathogen that affects different pinniped and mammalian species. This announcement reports the genome sequence of the type strain ATCC 51973 isolated in Norway from clinical specimens of harbor seal (Phoca vitulina), revealing interesting genes related to possible virulence factors. PMID:26586875

  15. Effect of Lactobacillus brevis ATCC 8287 as a feeding supplement on the performance and immune function of piglets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lactobacillus brevis ATCC 8287, a surface (S-layer) strain, possesses a variety of functional properties that make it both a potential probiotic and a good vaccine vector candidate. With this in mind, our aim was to study the survival of L. brevis in the porcine gut and investigate the effect of th...

  16. Complete Genome Sequence of Gluconacetobacter hansenii Strain NQ5 (ATCC 53582), an Efficient Producer of Bacterial Cellulose.

    PubMed

    Pfeffer, Sarah; Mehta, Kalpa; Brown, R Malcolm

    2016-01-01

    This study reports the release of the complete nucleotide sequence of Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis. PMID:27516505

  17. Genome sequence alterations detected upon passage of Burkholderia mallei ATCC 23344 in culture and in mammalian hosts

    PubMed Central

    Romero, Claudia M; DeShazer, David; Feldblyum, Tamara; Ravel, Jacques; Woods, Donald; Kim, H Stanley; Yu, Yan; Ronning, Catherine M; Nierman, William C

    2006-01-01

    Background More than 12,000 simple sequence repeats (SSRs) have been identified in the genome of Burkholderia mallei ATCC 23344. As a demonstrated mechanism of phase variation in other pathogenic bacteria, these may function as mutable loci leading to altered protein expression or structure variation. To determine if such alterations are occurring in vivo, the genomes of various single-colony passaged B. mallei ATCC 23344 isolates, one from each source, were sequenced from culture, a mouse, a horse, and two isolates from a single human patient, and the sequence compared to the published B. mallei ATCC 23344 genome sequence. Results Forty-nine insertions and deletions (indels) were detected at SSRs in the five passaged strains, a majority of which (67.3%) were located within noncoding areas, suggesting that such regions are more tolerant of sequence alterations. Expression profiling of the two human passaged isolates compared to the strain before passage revealed alterations in the mRNA levels of multiple genes when grown in culture. Conclusion These data support the notion that genome variability upon passage is a feature of B. mallei ATCC23344, and that within a host B. mallei generates a diverse population of clones that accumulate genome sequence variation at SSR and other loci. PMID:16953889

  18. Complete Genome Sequence of Gluconacetobacter hansenii Strain NQ5 (ATCC 53582), an Efficient Producer of Bacterial Cellulose.

    PubMed

    Pfeffer, Sarah; Mehta, Kalpa; Brown, R Malcolm

    2016-08-11

    This study reports the release of the complete nucleotide sequence of Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis.

  19. Complete Genome Sequence of Gluconacetobacter hansenii Strain NQ5 (ATCC 53582), an Efficient Producer of Bacterial Cellulose

    PubMed Central

    Pfeffer, Sarah; Mehta, Kalpa

    2016-01-01

    This study reports the release of the complete nucleotide sequence of Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis. PMID:27516505

  20. Meta-analysis: Lactobacillus reuteri strain DSM 17938 (and the original strain ATCC 55730) for treating acute gastroenteritis in children.

    PubMed

    Szajewska, H; Urbańska, M; Chmielewska, A; Weizman, Z; Shamir, R

    2014-09-01

    Lactobacillus reuteri ATCC 55730 has been shown to provide a moderate clinical effect in the treatment of acute gastroenteritis (AGE) in children. However, as the L. reuteri ATCC 55730 strain was found to carry potentially transferable resistance traits for tetracycline and lincomycin, it was replaced by a new strain, L. reuteri DSM 17938, without unwanted plasmid-borne antibiotic resistance. Bioequivalence of the two strains has been suggested. We aimed to systematically evaluate data on the effectiveness of L. reuteri DSM 17938 and the original strain, L. reuteri ATCC 55730, in the treatment of AGE in children. The Cochrane Library, MEDLINE, and EMBASE databases, reference lists, and abstract books of major scientific meetings were searched in August 2013, with no language restrictions, for relevant randomised controlled trials (RCTs). Two RCTs (n=196) that evaluated L. reuteri DSM 17938 and three RCTs (n=156) that evaluated L. reuteri ATCC 55730, which involved hospitalised children aged 3 to 60 months, met the inclusion criteria. Compared with placebo or no treatment, DSM 17938 significantly reduced the duration of diarrhoea (mean difference -32 h, 95% confidence interval (CI): -41 to -24) and increased the chance of cure on day 3 (relative risk: 3.5, 95% CI: 1.2 to 10.8, random effects model). Similar results were obtained with the original strain, L. reuteri ATCC 55730. In conclusion, in hospitalised children, use of both strains of L. reuteri reduced the duration of diarrhoea, and more children were cured within 3 days. Data from outpatients and countryspecific cost-effectiveness analyses are needed. Given the limited data and the methodological limitations of the included trials, the evidence should be viewed with caution.

  1. In situ hydrogen, acetone, butanol, ethanol and microdiesel production by Clostridium acetobutylicum ATCC 824 from oleaginous fungal biomass.

    PubMed

    Hassan, Elhagag Ahmed; Abd-Alla, Mohamed Hemida; Bagy, Magdy Mohamed Khalil; Morsy, Fatthy Mohamed

    2015-08-01

    An in situ batch fermentation technique was employed for biohydrogen, acetone, butanol, ethanol and microdiesel production from oleaginous fungal biomass using the anaerobic fermentative bacterium Clostridium acetobutylicum ATCC 824. Oleaginous fungal Cunninghamella echinulata biomass which has ability to accumulate up to 71% cellular lipid was used as the substrate carbon source. The maximum cumulative hydrogen by C. acetobutylicum ATCC 824 from crude C. echinulata biomass was 260 ml H2 l(-1), hydrogen production efficiency was 0.32 mol H2 mole(-1) glucose and the hydrogen production rate was 5.2 ml H2 h(-1). Subsequently, the produced acids (acetic and butyric acids) during acidogenesis phase are re-utilized by ABE-producing clostridia and converted into acetone, butanol, and ethanol. The total ABE produced by C. acetobutylicum ATCC 824 during batch fermentation was 3.6 g l(-1) from crude fungal biomass including acetone (1.05 g l(-1)), butanol (2.19 g l(-1)) and ethanol (0.36 g l(-1)). C. acetobutylicum ATCC 824 has ability to produce lipolytic enzymes with a specific activity 5.59 U/mg protein to hydrolyze ester containing substrates. The lipolytic potential of C. acetobutylicum ATCC 824 was used as a biocatalyst for a lipase transesterification process using the produced ethanol from ABE fermentation for microdiesel production. The fatty acid ethyl esters (microdiesel) generated from the lipase transesterification of crude C. echinulata dry mass was analyzed by GC/MS as 15.4% of total FAEEs. The gross energy content of biohydrogen, acetone, butanol, ethanol and biodiesel generated through C. acetobutylicum fermentation from crude C. echinulata dry mass was 3113.14 kJ mol(-1). These results suggest a possibility of integrating biohydrogen, acetone, butanol and ethanol production technology by C. acetobutylicum with microdiesel production from crude C. echinulata dry mass and therefore improve the feasibility and commercialization of bioenergy production.

  2. Investigation of the Amycolatopsis sp. Strain ATCC 39116 Vanillin Dehydrogenase and Its Impact on the Biotechnical Production of Vanillin

    PubMed Central

    Fleige, Christian; Hansen, Gunda; Kroll, Jens

    2013-01-01

    The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDHATCC 39116). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDHATCC 39116 was purified to apparent electrophoretic homogeneity and exhibited NAD+-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Kmr mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Kmr mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin. PMID:23064333

  3. In situ hydrogen, acetone, butanol, ethanol and microdiesel production by Clostridium acetobutylicum ATCC 824 from oleaginous fungal biomass.

    PubMed

    Hassan, Elhagag Ahmed; Abd-Alla, Mohamed Hemida; Bagy, Magdy Mohamed Khalil; Morsy, Fatthy Mohamed

    2015-08-01

    An in situ batch fermentation technique was employed for biohydrogen, acetone, butanol, ethanol and microdiesel production from oleaginous fungal biomass using the anaerobic fermentative bacterium Clostridium acetobutylicum ATCC 824. Oleaginous fungal Cunninghamella echinulata biomass which has ability to accumulate up to 71% cellular lipid was used as the substrate carbon source. The maximum cumulative hydrogen by C. acetobutylicum ATCC 824 from crude C. echinulata biomass was 260 ml H2 l(-1), hydrogen production efficiency was 0.32 mol H2 mole(-1) glucose and the hydrogen production rate was 5.2 ml H2 h(-1). Subsequently, the produced acids (acetic and butyric acids) during acidogenesis phase are re-utilized by ABE-producing clostridia and converted into acetone, butanol, and ethanol. The total ABE produced by C. acetobutylicum ATCC 824 during batch fermentation was 3.6 g l(-1) from crude fungal biomass including acetone (1.05 g l(-1)), butanol (2.19 g l(-1)) and ethanol (0.36 g l(-1)). C. acetobutylicum ATCC 824 has ability to produce lipolytic enzymes with a specific activity 5.59 U/mg protein to hydrolyze ester containing substrates. The lipolytic potential of C. acetobutylicum ATCC 824 was used as a biocatalyst for a lipase transesterification process using the produced ethanol from ABE fermentation for microdiesel production. The fatty acid ethyl esters (microdiesel) generated from the lipase transesterification of crude C. echinulata dry mass was analyzed by GC/MS as 15.4% of total FAEEs. The gross energy content of biohydrogen, acetone, butanol, ethanol and biodiesel generated through C. acetobutylicum fermentation from crude C. echinulata dry mass was 3113.14 kJ mol(-1). These results suggest a possibility of integrating biohydrogen, acetone, butanol and ethanol production technology by C. acetobutylicum with microdiesel production from crude C. echinulata dry mass and therefore improve the feasibility and commercialization of bioenergy production. PMID

  4. Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model.

    PubMed

    Seo, Ki-Weon; Kim, Sae-Hae; Park, Jisang; Son, Youngok; Yoo, Han Sang; Lee, Kyung-Yeol; Jang, Yong-Suk

    2013-01-15

    Actinobacillus pleuropneumoniae is an infective agent that leads to porcine pleuropneumonia, a disease that causes severe economic losses in the swine industry. Based on the fact that the respiratory tract is the primary site for bacterial infection, it has been suggested that bacterial exclusion in the respiratory tract through mucosal immune induction is the most effective disease prevention strategy. ApxIIA is a vaccine candidate against A. pleuropneumoniae infection, and fragment #5 (aa. 439-801) of ApxIIA contains the major epitopes for effective vaccination. In this study, we used mice to verify the efficacy of intranasal immunization with fragment #5 in the induction of protective immunity against nasal challenge with A. pleuropneumoniae and compared its efficacy with that of subcutaneous immunization. Intranasal immunization of the fragment induced significantly higher systemic and mucosal immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. Intranasal immunization not only efficiently inhibited the bacterial colonization in respiratory organs, but also prevented alveolar tissue damage in infectious condition similar to that of a contaminated pig. Moreover, intranasal immunization with fragment #5 provided acquired protective immunity against intranasal challenge with A. pleuropneumoniae serotype 2. In addition, it conferred cross-protection against serotype 5, a heterologous pathogen that causes severe disease by ApxI and ApxII secretion. Collectively, intranasal immunization with fragment #5 of ApxIIA can be considered an efficient protective immunization procedure against A. pleuropneumoniae infection.

  5. Analytical performance of an immunologic-based periodontal bacterial test for simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.

    PubMed

    Snyder, B; Ryerson, C C; Corona, H; Grogan, E A; Reynolds, H S; Contestable, P B; Boyer, B P; Mayer, J; Mangan, T; Norkus, N; Zambon, J J; Genco, R J

    1996-05-01

    The analytical performance of a membrane-based immunoassay for the simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia (including Prevotella nigrescens) was investigated. Positive reactions were observed for 71 of 71 reference strains and recent oral isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. No cross-reactivity was observed with 39 other common oral and environmental species. The specificity of the test was unaffected by the presence of potential oral interferents including whole blood, white blood cells, mucin, saliva, toothpastes, and oral rinses. A proficiency test by dental professionals using a standardized set of unknown simulated samples yielded a sensitivity of 97% (116/120) and a 100% specificity (240/ 240). An additional group including dental professionals and high school students was shown to be 99% proficient (1385/1397) in distinguishing proper from improper test function when processing control samples with normal test devices and devices with simulated error conditions. Comparisons to a culture standard for 104 subgingival plaque samples collected from 26 adult periodontitis patients yielded > 98% specificity for each of the test bacteria. In addition, the detection threshold for the test was determined to be equivalent to 10(4) cultivable test bacteria when compared to the culture standard. The data indicate that this membrane immunoassay is a valid and easy-to-use method for the detection of A. actinomycetemcomitans, P. gingivalis, and P. intermedia in subgingival plaque, at levels above the detection threshold of the test.

  6. The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin

    PubMed Central

    Choi, Kyoung-Jae; Grass, Susan; Paek, Seonghee; St. Geme, Joseph W.; Yeo, Hye-Jeong

    2010-01-01

    The Haemophilus influenzae HMW1 adhesin is an important virulence exoprotein that is secreted via the two-partner secretion pathway and is glycosylated at multiple asparagine residues in consensus N-linked sequons. Unlike the heavily branched glycans found in eukaryotic N-linked glycoproteins, the modifying glycan structures in HMW1 are mono-hexoses or di-hexoses. Recent work demonstrated that the H. influenzae HMW1C protein is the glycosyltransferase responsible for transferring glucose and galactose to the acceptor sites of HMW1. An Actinobacillus pleuropneumoniae protein designated ApHMW1C shares high-level homology with HMW1C and has been assigned to the GT41 family, which otherwise contains only O-glycosyltransferases. In this study, we demonstrated that ApHMW1C has N-glycosyltransferase activity and is able to transfer glucose and galactose to known asparagine sites in HMW1. In addition, we found that ApHMW1C is able to complement a deficiency of HMW1C and mediate HMW1 glycosylation and adhesive activity in whole bacteria. Initial structure-function studies suggested that ApHMW1C consists of two domains, including a 15-kDa N-terminal domain and a 55-kDa C-terminal domain harboring glycosyltransferase activity. These findings suggest a new subfamily of HMW1C-like glycosyltransferases distinct from other GT41 family O-glycosyltransferases. PMID:21209858

  7. Oral and aerosol immunization with viable or inactivated Actinobacillus pleuropneumoniae bacteria: antibody response to capsular polysaccharides in bronchoalveolar lavage fluids (BALF) and sera of pigs.

    PubMed

    Hensel, A; Pabst, R; Bunka, S; Petzoldt, K

    1994-04-01

    To investigate the antibody response after local application of lung-pathogenic bacteria, pigs were immunized with viable or inactivated Actinobacillus pleuropneumoniae by the oral and aerogenous route. After 3 weeks class-specific immunoglobulins against purified A. pleuropneumoniae capsular polysaccharides (CP) were determined in serum and BALF by ELISA. A significant increase of IgA antibodies was found in BALF but not in sera of all immunized pigs. Oral immunization with viable A. pleuropneumoniae and aerosol immunization with either viable or inactivated bacteria resulted in a significant increase of IgG antibodies to the CP antigen in BALF, whereas only aerosol exposure to viable bacteria resulted in a significant increase in IgG antibodies in serum. A significant increase in anti-CP IgM in BALF was observed after aerosol exposure but not after oral immunization. IgM antibodies towards CP increased significantly by both routes of immunization with viable bacteria. The anti-CP activity of all three isotypes in sera and BALF was low in all groups compared with the positive controls, although inoculation of viable A. pleuropneumoniae led to higher levels of antibody concentration than inactivated bacteria. Our results indicate a traffic of primed lymphocytes from the gut into the bronchoalveolar airways and further support the hypothesis that polysaccharide-specific B cells may functionally mature at the mucosal surfaces.

  8. Pigs aerogenously immunized with genetically inactivated (ghosts) or irradiated Actinobacillus pleuropneumoniae are protected against a homologous aerosol challenge despite differing in pulmonary cellular and antibody responses.

    PubMed

    Katinger, A; Lubitz, W; Szostak, M P; Stadler, M; Klein, R; Indra, A; Huter, V; Hensel, A

    1999-08-20

    Aerosol immunization is a safe way to induce complete protection against pleuropneumonia in pigs caused by the lung pathogenic bacterium Actinobacillus pleuropneumoniae. In order to determine the local immune responses of vaccinees in concomitant with protection, lung lining fluid before and 3 weeks after immunization from pigs immunized three times with aerosols of either genetically inactivated ghosts which represent whole cell envelope preparations, or irradiated bacteria were examined following an homologous aerosol challenge. Specific antibody isotypes in the bronchoalveolar lavage were assayed by whole cell ELISAs. Total and relative numbers of cells including lymphocyte subsets were determined. In both vaccinated groups a net influx of plasma cells and lymphocytes, as well as a significant increase of specific IgG occurred. Concurrently, the CD4+/CD8+ ratio was found to increase after aerosol immunization. The lymphocyte subsets of IgG+ and IgA+ cells were found significantly higher in the group immunized with irradiated bacteria when compared to pigs immunized with bacterial ghosts. The latter group showed a significant increase of IgA, IgM, and a net influx of lymphoid blasts and granulocytes in the bronchoalveolar lining fluid. Although differences between the local immune responses of both immunized groups occurred, a significant increase of specific IgG and a net influx of plasma cells and lymphocytes were found to be associated with complete protection against a homologous aerosol challenge infection.

  9. Genetic and antigenic characteristics of ApxIIA and ApxIIIA from Actinobacillus pleuropneumoniae serovars 2, 3, 4, 6, 8 and 15.

    PubMed

    To, Ho; Nagai, Shinya; Iwata, Akira; Koyama, Tomohiro; Oshima, Atsushi; Tsutsumi, Nobuyuki

    2016-07-01

    Apx toxins produced by Actinobacillus pleuropneumoniae are essential components of new generation vaccines. In this study, apxIIA and apxIIIA genes of serovars 2, 3, 4, 6, 8 and 15 were cloned and sequenced. Amino acid sequences of ApxIIA proteins of serovars 2, 3, 4, 6, 8 and 15 were almost identical to those of serovars 1, 5, 7, 9 and 11-13. Immunoblot analysis showed that rApxIIA from serovars 2 and 15 reacts strongly with sera from animals infected with various serovars. Sequence analysis revealed that ApxIIIA proteins has two variants, one in strains of serovar 2 and the other in strains of serovars 3, 4, 6, 8 and 15. A mouse cross-protection study showed that mice actively immunized with rApxIIIA/2 or rApxIIIA/15 are protected against challenge with A. pleuropneumoniae strains of serovars 3, 4, 6, 8, 15, and 2 expressing ApxIII/15 and ApxIII/2, respectively. Similarly, mice passively immunized with rabbit anti-rApxIIIA/2 or anti-rApxIIIA/15 sera were found to be protected against challenge with strains of serovars 2 and 15. Our study revealed antigenic and sequence similarities within ApxIIA and ApxIIIA proteins, which may help in the development of effective vaccines against disease caused by A. pleuropneumoniae.

  10. Pharmacokinetic/pharmacodynamic evaluation of marbofloxacin in the treatment of Haemophilus parasuis and Actinobacillus pleuropneumoniae infections in nursery and fattener pigs using Monte Carlo simulations.

    PubMed

    Vilalta, C; Giboin, H; Schneider, M; El Garch, F; Fraile, L

    2014-12-01

    This study evaluated the theoretical clinical outcome of three marbofloxacin posology regimens in two groups of pigs (weaners and fatteners) for the treatment of Actinobacillus pleuropneumoniae (App) and Haemophilus parasuis (Hp) infection and the appearance of resistant bacteria due to the antibiotic treatment. The probability of target attainment (PTA) for pharmacokinetic/pharmacodynamics (PK/PD) ratios associated with clinical efficacy and with the appearance of antimicrobial resistance for fluoroquinolones at each minimum inhibitory concentration (MIC) or mutant prevention concentration (MPC) were calculated, respectively. The cumulative fraction of response (CFR) was calculated for the three posology regimens against App and they ranged from 91.12% to 96.37% in weaners and from 93% to 97.43% in fatteners, respectively. In the case of Hp, they ranged from 80.52% to 85.14% in weaners and from 82.01% to 88.49% in fatteners, respectively. Regarding the PTA of the PK/PD threshold associated with the appearance of antimicrobial resistance, results showed that marbofloxacin would prevent resistances in most of the animals up to the MPC value of 1 μg/mL.

  11. Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release.

    PubMed

    Wang, Lei; Qin, Wanhai; Zhang, Jing; Bao, Chuntong; Zhang, Hu; Che, Yanyi; Sun, Changjiang; Gu, Jingmin; Feng, Xin; Du, Chongtao; Han, Wenyu; Richard, Paul Langford; Lei, Liancheng

    2016-04-05

    Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs.

  12. Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release

    PubMed Central

    Wang, Lei; Qin, Wanhai; Zhang, Jing; Bao, Chuntong; Zhang, Hu; Che, Yanyi; Sun, Changjiang; Gu, Jingmin; Feng, Xin; Du, Chongtao; Han, Wenyu; Richard, Paul Langford; Lei, Liancheng

    2016-01-01

    Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs. PMID:27046446

  13. Cloning and characterization of the gene coding for NADPH-sulfite reductase hemoprotein from Actinobacillus pleuropneumoniae and use of the protein product as a vaccine.

    PubMed

    Willson, P J; Gerlach, G F; Klashinsky, S; Potter, A A

    2001-10-01

    An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate and screened with serum produced in pigs that had been vaccinated with the anionic fraction of a sodium chloride extract. One E. coli transformant was isolated that produced a large amount of a protein with an electrophoretic mobility of about 67,000 molecular mass. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by restriction enzyme digestion and nucleotide sequence analysis which showed strong homology with the cysI gene of E. coli. One open reading frame of 1764 bases in length was detected which encoded a cysI protein from serotype 1, with a calculated molecular mass of 66,678. The DNA encoding the protein was labeled with radio-isotope and the homologous gene was isolated from an A. pleuropneumoniae serotype 5a library. The serotype 5a gene was the same length, but the cysI protein from serotype 5a was slightly larger (66,849) due to 8 substitutions in the amino acid sequence. Expression plasmids containing cysI from either serotype of A. pleuropneumoniae complemented an E. coli cysI mutant. Pigs vaccinated with the recombinant cysI were protected from challenge with A. pleuropneumoniae of the homologous serotype.

  14. Magnetic beads-based enzymatic spectrofluorometric assay for rapid and sensitive detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae.

    PubMed

    Wei, Bo; Li, Fang; Yang, Huicui; Yu, Lei; Zhao, Kaihong; Zhou, Rui; Hu, Yonggang

    2012-05-15

    In this paper, a simple, easily-operated and enzyme-amplified fluorescence immunoassay method using magnetic particles for the detection of antibody against Actinobacillus pleuropneumoniae (APP) has been presented. The A protein of APP Repeats-in-Toxin IV (ApxIVA) with high specificity to the APP species was immobilized onto the magnetic bead surfaces. Horseradish peroxidase (HRP), which can catalyze the substrate 4-hydroxyphenylacetic acid (p-HPA), generating fluorescent bi-p, p'-hydroxyphenylacetic acid (DBDA), was selected as an enzymatic-amplified tracer. The ApxIVA antibody was detected for the presence of APP infection by measuring the fluorescence intensity of DBDA. Under optimal conditions, the calibration plot obtained for standard positive serum was approximately linear within the dilution range 1:160-1:5120. The limit of detection (LOD) for the assay was 1:10240, considerably lower than that of ApxIVA-ELISA (1:320) (S/N=3). A series of repeatability measurements of using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation (RSD) of 4.8% (n=11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor yielded an efficiency of 89.7%, sensitivity of 90.9% and specificity of 89.3% compared with ApxIVA-ELISA.

  15. Azotobacter Genomes: The Genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412)

    PubMed Central

    Robson, Robert L.; Jones, Robert; Robson, R. Moyra; Schwartz, Ariel; Richardson, Toby H.

    2015-01-01

    The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these

  16. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    SciTech Connect

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-07-29

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  17. Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

    PubMed Central

    Lo Grasso, Letizia; Maffioli, Sonia; Sosio, Margherita; Bibb, Mervyn; Puglia, Anna Maria

    2015-01-01

    ABSTRACT The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, overexpression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, and dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, and dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation. IMPORTANCE This report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis

  18. Effect of Low Shear Modeled Microgravity (LSMMG) on the Probiotic Lactobacillus Acidophilus ATCC 4356

    NASA Technical Reports Server (NTRS)

    Stahl, S.; Voorhies, A.; Lorenzi, H.; Castro-Wallace, S.; Douglas, G.

    2016-01-01

    The introduction of generally recognized as safe (GRAS) probiotic microbes into the spaceflight food system has the potential for use as a safe, non-invasive, daily countermeasure to crew microbiome and immune dysregulation. However, the microgravity effects on the stress tolerances and genetic expression of probiotic bacteria must be determined to confirm translation of strain benefits and to identify potential for optimization of growth, survival, and strain selection for spaceflight. The work presented here demonstrates the translation of characteristics of a GRAS probiotic bacteria to a microgravity analog environment. Lactobacillus acidophilus ATCC 4356 was grown in the low shear modeled microgravity (LSMMG) orientation and the control orientation in the rotating wall vessel (RWV) to determine the effect of LSMMG on the growth, survival through stress challenge, and gene expression of the strain. No differences were observed between the LSMMG and control grown L. acidophilus, suggesting that the strain will behave similarly in spaceflight and may be expected to confer Earth-based benefits.

  19. Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production.

    PubMed

    Chen, Cheng; Li, Yanyan; Hu, Jinyu; Dong, Xunyan; Wang, Xiaoyuan

    2015-05-01

    In this study, an L-valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE, alaT and ilvA combined with the overexpression of six genes ilvB, ilvN, ilvC, lrp1, brnF and brnE. Overexpression of lrp1 alone increased L-valine production by 16-fold. Deletion of the aceE, alaT and ilvA increased L-valine production by 44-fold. Overexpression of the six genes ilvB, ilvN, ilvC, lrp1, brnE and brnF in the triple deletion mutant WCC003 further increased L-valine production. The strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE produced 243mM L-valine in flask cultivation and 437mM (51g/L) L-valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l-alanine and l-isoleucine that are usually found in the fermentation of L-valine-producing C. glutamicum.

  20. Sophorolipids Production by Candida bombicola ATCC 22214 and its Potential Application in Microbial Enhanced Oil Recovery.

    PubMed

    Elshafie, Abdulkadir E; Joshi, Sanket J; Al-Wahaibi, Yahya M; Al-Bemani, Ali S; Al-Bahry, Saif N; Al-Maqbali, Dua'a; Banat, Ibrahim M

    2015-01-01

    Biosurfactant production using Candida bombicola ATCC 22214, its characterization and potential applications in enhancing oil recovery were studied at laboratory scale. The seed media and the production media were standardized for optimal growth and biosurfactant production. The production media were tested with different carbon sources: glucose (2%w/v) and corn oil (10%v/v) added separately or concurrently. The samples were collected at 24 h interval up to 120 h and checked for growth (OD660), and biosurfactant production [surface tension (ST) and interfacial tension (IFT)]. The medium with both glucose and corn oil gave better biosurfactant production and reduced both ST and IFT to 28.56 + 0.42mN/m and 2.13 + 0.09mN/m, respectively within 72 h. The produced biosurfactant was quite stable at 13-15% salinity, pH range of 2-12, and at temperature up to 100°C. It also produced stable emulsions (%E24) with different hydrocarbons (pentane, hexane, heptane, tridecane, tetradecane, hexadecane, 1-methylnaphthalene, 2,2,4,4,6,8-heptamethylnonane, light and heavy crude oil). The produced biosurfactant was extracted using ethyl acetate and characterized as a mixture of sophorolipids (SPLs). The potential of SPLs in enhancing oil recovery was tested using core-flooding experiments under reservoir conditions, where additional 27.27% of residual oil (Sor) was recovered. This confirmed the potential of SPLs for applications in microbial enhanced oil recovery.

  1. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

    PubMed

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-12-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  2. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

    PubMed Central

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-01-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant. PMID:24688492

  3. Isolation and characterisation of dipeptidyl peptidase IV from Prevotella loescheii ATCC 15930.

    PubMed

    Koreeda, Y; Hayakawa, M; Ikemi, T; Abiko, Y

    2001-08-01

    A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3.4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100 degrees C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two identical subunits and is folded in 2% SDS. The optimum pH, with glycyl-prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd(2+), Zn(2+), Hg(2+), Co(2+), and serine proteinase inhibitor di-isopropylfluorophosphate. PMID:11389867

  4. Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824.

    PubMed

    Yu, Yang; Tangney, Martin; Aass, Hans C; Mitchell, Wilfrid J

    2007-03-01

    Although the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolize a wide range of carbohydrates offers the potential for revival based on the use of cheap, low-grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolyzed by a phospho-beta-galactosidase. These activities are induced during growth on lactose but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho-beta-galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed. PMID:17209069

  5. Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli

    PubMed Central

    Jaramillo, Michael L; Abanto, Michel; Quispe, Ruth L; Calderón, Julio; del Valle, Luís J; Talledo, Miguel; Ramírez, Pablo

    2012-01-01

    Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases. PMID:23055613

  6. Staphylococcus saprophyticus ATCC 15305 is internalized into human urinary bladder carcinoma cell line 5637.

    PubMed

    Szabados, Florian; Kleine, Britta; Anders, Agnes; Kaase, Martin; Sakinç, Türkân; Schmitz, Inge; Gatermann, Sören

    2008-08-01

    Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.

  7. Sulphate production by Paracoccus pantotrophus ATCC 35512 from different sulphur substrates: sodium thiosulphate, sulphite and sulphide.

    PubMed

    Meyer, Daniel Derrossi; Andrino, Felipe Gabriel; Possedente de Lira, Simone; Fornaro, Adalgiza; Corção, Gertrudes; Brandelli, Adriano

    2016-01-01

    One of the problems in waste water treatment plants (WWTPs) is the increase in emissions of hydrogen sulphide (H2S), which can cause damage to the health of human populations and ecosystems. To control emissions of this gas, sulphur-oxidizing bacteria can be used to convert H2S to sulphate. In this work, sulphate detection was performed by spectrophotometry, ion chromatography and atomic absorption spectrometry, using Paracoccus pantotrophus ATCC 35512 as a reference strain growing in an inorganic broth supplemented with sodium thiosulphate (Na2S2O3·5H2O), sodium sulphide (Na2S) or sodium sulphite (Na2SO3), separately. The strain was metabolically competent in sulphate production. However, it was only possible to observe significant differences in sulphate production compared to abiotic control when the inorganic medium was supplemented with sodium thiosulphate. The three methods for sulphate detection showed similar patterns, although the chromatographic method was the most sensitive for this study. This strain can be used as a reference for sulphate production in studies with sulphur-oxidizing bacteria originating from environmental samples of WWTPs.

  8. Bismuth(III) deferiprone effectively inhibits growth of Desulfovibrio desulfuricans ATCC 27774.

    PubMed

    Barton, Larry L; Lyle, Daniel A; Ritz, Nathaniel L; Granat, Alex S; Khurshid, Ali N; Kherbik, Nada; Hider, Robert; Lin, Henry C

    2016-04-01

    Sulfate-reducing bacteria have been implicated in inflammatory bowel diseases and ulcerative colitis in humans and there is an interest in inhibiting the growth of these sulfide-producing bacteria. This research explores the use of several chelators of bismuth to determine the most effective chelator to inhibit the growth of sulfate-reducing bacteria. For our studies, Desulfovibrio desulfuricans ATCC 27774 was grown with nitrate as the electron acceptor and chelated bismuth compounds were added to test for inhibition of growth. Varying levels of inhibition were attributed to bismuth chelated with subsalicylate or citrate but the most effective inhibition of growth by D. desulfuricans was with bismuth chelated by deferiprone, 3-hydroxy-1,2-dimethyl-4(1H)-pyridone. Growth of D. desulfuricans was inhibited by 10 μM bismuth as deferiprone:bismuth with either nitrate or sulfate respiration. Our studies indicate deferiprone:bismuth has bacteriostatic activity on D. desulfuricans because the inhibition can be reversed following exposure to 1 mM bismuth for 1 h at 32 °C. We suggest that deferiprone is an appropriate chelator for bismuth to control growth of sulfate-reducing bacteria because deferiprone is relatively nontoxic to animals, including humans, and has been used for many years to bind Fe(III) in the treatment of β-thalassemia. PMID:26896170

  9. Biosurfactant Production by Cultivation of Bacillus atrophaeus ATCC 9372 in Semidefined Glucose/Casein-Based Media

    NASA Astrophysics Data System (ADS)

    Das Neves, Luiz Carlos Martins; de Oliveira, Kátia Silva; Kobayashi, Márcio Junji; Vessoni Penna, Thereza Christina; Converti, Attilio

    Biosurfactants are proteins with detergent, emulsifier, and antimicrobial actions that have potential application in environmental applications such as the treatment of organic pollutants and oil recovery. Bacillus atrophaeus strains are nonpathogenic and are suitable source of biosurfactants, among which is surfactin. The aim of this work is to establish a culture medium composition able to stimulate biosurfactants production by B. atrophaeus ATCC 9372. Batch cultivations were carried out in a rotary shaker at 150 rpm and 35°C for 24 h on glucose- and/or casein-based semidefined culture media also containing sodium chloride, dibasic sodium phosphate, and soy flour. The addition of 14.0 g/L glucose in a culture medium containing 10.0 g/L of casein resulted in 17 times higher biosurfactant production (B max=635.0 mg/L). Besides, the simultaneous presence of digested casein (10.0 g/L), digested soy flour (3.0 g/L), and glucose (18.0 g/L) in the medium was responsible for a diauxic effect during cell growth. Once the diauxie started, the average biosurfactants concentration was 16.8% less than that observed before this phenomenon. The capability of B. atrophaeus strain to adapt its own metabolism to use several nutrients as energy sources and to preserve high levels of biosurfactants in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

  10. Evaluation of Cyanothece sp. ATCC 51142 as a candidate for inclusion in a CELSS.

    PubMed

    Schneegurt, M A; Arieli, B; Nielsen, S S; Trumbo, P R; Sherman, L A

    1996-01-01

    Controlled ecological life support systems (CELSS) have been proposed to make long-duration manned space flights more cost-effective. Higher plants will presumably provide food and a breathable atmosphere for the crew. It has been suggested that imbalances between the CO2/O2 gas exchange ratios of the heterotrophic and autotrophic components of the system will inevitably lead to an unstable system, and the loss of O2 from the atmosphere. Ratio imbalances may be corrected by including a second autotroph with an appropriate CO2/O2 gas exchange ratio. Cyanothece sp. ATCC 51142 is a large unicellular N2-fixing cyanobacterium, exhibiting high growth rates under diverse physiological conditions. A rat-feeding study showed the biomass to be edible. Furthermore, it may have a CO2/O2 gas exchange ratio that theoretically can compensate for ratio imbalances. It is suggested that Cyanothece spp. could fulfill several roles in a CELSS: supplementing atmosphere recycling, generating fixed N from the air, providing a balanced protein supplement, and protecting a CELSS in case of catastrophic crop failure.

  11. Evaluation of Cyanothece sp. ATCC 51142 as a candidate for inclusion in a CELSS

    NASA Technical Reports Server (NTRS)

    Schneegurt, M. A.; Arieli, B.; Nielsen, S. S.; Trumbo, P. R.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

    1996-01-01

    Controlled ecological life support systems (CELSS) have been proposed to make long-duration manned space flights more cost-effective. Higher plants will presumably provide food and a breathable atmosphere for the crew. It has been suggested that imbalances between the CO2/O2 gas exchange ratios of the heterotrophic and autotrophic components of the system will inevitably lead to an unstable system, and the loss of O2 from the atmosphere. Ratio imbalances may be corrected by including a second autotroph with an appropriate CO2/O2 gas exchange ratio. Cyanothece sp. ATCC 51142 is a large unicellular N2-fixing cyanobacterium, exhibiting high growth rates under diverse physiological conditions. A rat-feeding study showed the biomass to be edible. Furthermore, it may have a CO2/O2 gas exchange ratio that theoretically can compensate for ratio imbalances. It is suggested that Cyanothece spp. could fulfill several roles in a CELSS: supplementing atmosphere recycling, generating fixed N from the air, providing a balanced protein supplement, and protecting a CELSS in case of catastrophic crop failure.

  12. Listeria ivanovii ATCC 19119 strain behaviour is modulated by iron and acid stress.

    PubMed

    Longhi, Catia; Ammendolia, Maria Grazia; Conte, Maria Pia; Seganti, Lucilla; Iosi, Francesca; Superti, Fabiana

    2014-09-01

    It has been suggested that the rarity of human listeriosis due to Listeria ivanovii reflects not only host tropism factors but also the rare occurrence of this species in the environment, compared with Listeria monocytogenes. In the present study we evaluate the effects on the reference strain L. ivanovii ATCC 19119 behaviour of two combined stresses, low iron availability and acid environment, that bacteria can encounter in the passage from saprophytic life to the host. In these conditions, L. ivanovii evidenced a different behaviour compared to L. monocytogenes exposed to similar conditions. L. ivanovii was not able to mount an acid tolerance response (ATR) even if, upon entry into the stationary phase in iron-loaded medium, growth phase-dependent acid resistance (AR) was evidenced. Moreover, bacteria grown in iron excess and acidic pH showed the higher invasion value in Caco-2 cells, even though it was not able to efficiently multiply. On the contrary, low iron and acidic conditions improved invasion ability in amniotic WISH cells.

  13. Production of single cell oil from Lipomyces starkeyi ATCC 56304 using biorefinery by-products.

    PubMed

    Probst, Kyle V; Vadlani, Praveen V

    2015-12-01

    Single cell oil (SCO) is a valuable noncrop-based renewable oil source. Hemicellulose derived sugars can be utilized to produce SCO using the oleaginous yeast Lipomyces starkeyi ATCC 56304. Bran by-products were tested as hemicellulose-rich feedstocks for the production of SCO. Whole and destarched corn and wheat bran hydrolysates were produced using hydrothermal and dilute sulfuric acid (0%, 0.5%, 1.0%, v/v) pretreatment along with enzymatic hydrolysis. Whole bran hydrolysates produced from hydrothermal pretreatment generated the highest average oil yields of 126.7 and 124.3 mg oil/g sugar for both wheat and corn bran, respectively. 1.0% acid pretreatment was effective for the destarched bran generating a hemicellulose hydrolysis efficiency of 94% and 84% for wheat and corn bran, respectively, resulting in the highest oil yield of 70.7 mg oil/g sugar. The results indicate pretreated corn and wheat bran hydrolysates can serve as viable feedstocks for oleaginous yeast SCO bioconversion.

  14. Transcriptional and Mutational Analysis of the Uptake Hydrogenase of the Filamentous Cyanobacterium Anabaena variabilis ATCC 29413

    PubMed Central

    Happe, Thomas; Schütz, Kathrin; Böhme, Herbert

    2000-01-01

    A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase (hupSL) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation of A. variabilis. The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N2-fixing conditions, the mutant strain exhibited significantly increased rates in H2 accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H2 developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type. PMID:10692368

  15. Production of polyhydroxyalkanoates by Burkholderia cepacia ATCC 17759 using a detoxified sugar maple hemicellulosic hydrolysate.

    PubMed

    Pan, Wenyang; Perrotta, Joseph A; Stipanovic, Arthur J; Nomura, Christopher T; Nakas, James P

    2012-03-01

    Sugar maple hemicellulosic hydrolysate containing 71.9 g/l of xylose was used as an inexpensive feedstock to produce polyhydroxyalkanoates (PHAs) by Burkholderia cepacia ATCC 17759. Several inhibitory compounds present in wood hydrolysate were analyzed for effects on cell growth and PHA production with strong inhibition observed at concentrations of 1 g/l furfural, 2 g/l vanillin, 7 g/l levulinic acid, and 1 M acetic acid. Gradual catabolism of lower concentrations of these inhibitors was observed in this study. To increase the fermentability of wood hydrolysate, several detoxification methods were tested. Overliming combined with low-temperature sterilization resulted in the highest removal of total inhibitory phenolics (65%). A fed-batch fermentation exhibited maximum PHA production after 96 h (8.72 g PHA/L broth and 51.4% of dry cell weight). Compositional analysis by NMR and physical-chemical characterization showed that PHA produced from wood hydrolysate was composed of polyhydroxybutyrate (PHB) with a molecular mass (M (N)) of 450.8 kDa, a melting temperature (T (m)) of 174.4°C, a glass transition temperature (T (g)) of 7.31°C, and a decomposition temperature (T (decomp)) of 268.6°C.

  16. Sophorolipids Production by Candida bombicola ATCC 22214 and its Potential Application in Microbial Enhanced Oil Recovery

    PubMed Central

    Elshafie, Abdulkadir E.; Joshi, Sanket J.; Al-Wahaibi, Yahya M.; Al-Bemani, Ali S.; Al-Bahry, Saif N.; Al-Maqbali, Dua’a; Banat, Ibrahim M.

    2015-01-01

    Biosurfactant production using Candida bombicola ATCC 22214, its characterization and potential applications in enhancing oil recovery were studied at laboratory scale. The seed media and the production media were standardized for optimal growth and biosurfactant production. The production media were tested with different carbon sources: glucose (2%w/v) and corn oil (10%v/v) added separately or concurrently. The samples were collected at 24 h interval up to 120 h and checked for growth (OD660), and biosurfactant production [surface tension (ST) and interfacial tension (IFT)]. The medium with both glucose and corn oil gave better biosurfactant production and reduced both ST and IFT to 28.56 + 0.42mN/m and 2.13 + 0.09mN/m, respectively within 72 h. The produced biosurfactant was quite stable at 13–15% salinity, pH range of 2–12, and at temperature up to 100°C. It also produced stable emulsions (%E24) with different hydrocarbons (pentane, hexane, heptane, tridecane, tetradecane, hexadecane, 1-methylnaphthalene, 2,2,4,4,6,8-heptamethylnonane, light and heavy crude oil). The produced biosurfactant was extracted using ethyl acetate and characterized as a mixture of sophorolipids (SPLs). The potential of SPLs in enhancing oil recovery was tested using core-flooding experiments under reservoir conditions, where additional 27.27% of residual oil (Sor) was recovered. This confirmed the potential of SPLs for applications in microbial enhanced oil recovery. PMID:26635782

  17. Proteome data to explore the impact of pBClin15 on Bacillus cereus ATCC 14579.

    PubMed

    Madeira, Jean-Paul; Alpha-Bazin, Béatrice; Armengaud, Jean; Omer, Hélène; Duport, Catherine

    2016-09-01

    This data article reports changes in the cellular and exoproteome of B. cereus cured from pBClin15.Time-course changes of proteins were assessed by high-throughput nanoLC-MS/MS. We report all the peptides and proteins identified and quantified in B. cereus with and without pBClin15. Proteins were classified into functional groups using the information available in the KEGG classification and we reported their abundance in term of normalized spectral abundance factor. The repertoire of experimentally confirmed proteins of B. cereus presented here is the largest ever reported, and provides new insights into the interplay between pBClin15 and its host B. cereus ATCC 14579. The data reported here is related to a published shotgun proteomics analysis regarding the role of pBClin15, "Deciphering the interactions between the Bacillus cereus linear plasmid, pBClin15, and its host by high-throughput comparative proteomics" Madeira et al. [1]. All the associated mass spectrometry data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (http://www.ebi.ac.uk/pride/), with the dataset identifier PRIDE: PXD001568, PRIDE: PXD002788 and PRIDE: PXD002789. PMID:27547804

  18. A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400.

    PubMed

    Gaballa, A; Koedam, N; Cornelis, P

    1996-08-01

    Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron-limiting conditions. A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) or zinc. This mutant was complemented by a 9.6 kb HindIII-BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH). The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins. By TnphoA mutagenesis and site-specific gene replacement it was found that the first three ORFs (cytA to cytC) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production. The presence of a putative haem-binding site in the CytA protein (WGSWWVWD) was confirmed. From analysis of a constructed phoA fusion, a periplasmic location was found for this motif. The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P. fluorescens. PMID:8878040

  19. Identification and characterization of the afsR homologue regulatory gene from Streptomyces peucetius ATCC 27952.

    PubMed

    Parajuli, Niranjan; Viet, Hung Trinh; Ishida, Kenji; Tong, Hang Thi; Lee, Hei Chan; Liou, Kwangkyoung; Sohng, Jae Kyung

    2005-01-01

    We have isolated an afsR homologue, called afsR-p, through genome analysis of Streptomyces peucetius ATCC 27952. AfsR-p shares 60% sequence identity with AfsR from Streptomyces coelicolor A3 (2). afsR-p was expressed under the control of the ermE* promoter in its hosts S. peucetius, Streptomyces lividans TK 24, Streptomyces clavuligerus and Streptomyces griseus. We observed overproduction of doxorubicin (4-fold) in S. peucetius, gamma-actinorhodin (2.6-fold) in S. lividans, clavulanic acid (1.5-fold) in S. clavuligerus and streptomycin (slight) in S. griseus. Overproduction was due to expression of the gene in these strains as compared to the wild-type strains harboring the vector only. Comparative study of the expression of afsR-p revealed that regulatory networking in Streptomyces is not uniform. We speculate that phosphorylated AfsR-p becomes bound to the promoter region of afsS. The latter activates other regulatory genes, including pathway regulatory genes, and induces the production of secondary metabolites including antibiotics. We identified specific conserved amino acids and exploited them for the isolation of the partial sequence of the afsR homologue from S. clavuligerus and Streptomyces achromogens (rubradirin producer). Such findings provide additional evidence for the presence of a serine/threonine and tyrosine kinase-dependent global regulatory network in Streptomyces.

  20. Biodesulfurization of a system containing synthetic fuel using Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, Danielle; de Oliveira, Débora; de Souza, Antônio A Ulson; Souza, Selene M A Guelli U

    2014-11-01

    The burning of fossil fuels has released a large quantity of pollutants into the atmosphere. In this context, sulfur dioxide is one of the most noxious gas which, on reacting with moist air, is transformed into sulfuric acid, causing the acid rain. In response, many countries have reformulated their legislation in order to enforce the commercialization of fuels with very low sulfur levels. The existing desulfurization processes cannot remove such low levels of sulfur and thus a biodesulfurization has been developed, where the degradation of sulfur occurs through the action of microorganisms. Rhodococcus erythropolis has been identified as one of the most promising bacteria for use in the biodesulfurization. In this study, the effectiveness of the strain R. erythropolis ATCC 4277 in the desulfurization of dibenzothiophene (DBT) was evaluated in a batch reactor using an organic phase (n-dodecane or diesel) concentrations of 20, 80, and 100 % (v/v). This strain was able to degrade 93.3, 98.0, and 95.5 % of the DBT in the presence of 20, 80, and 100 % (v/v) of dodecane, respectively. The highest value for the specific DBT degradation rate was 44 mmol DBT · kg DCW(-1) · h(-1), attained in the reactor containing 80 % (v/v) of n-dodecane as the organic phase.

  1. Elucidation of the metabolic pathway for dibenzothiophene desulphurization by Rhodococcus sp. strain IGTS8 (ATCC 53968).

    PubMed

    Oldfield, C; Pogrebinsky, O; Simmonds, J; Olson, E S; Kulpa, C F

    1997-09-01

    Rhodococcus sp. strain IGTS8 (ATCC 53968) is able to utilize dibenzothiophene (DBT) as a sole source of sulphur. The carbon skeleton of DBT is not metabolized and is conserved as 2-hydroxybiphenyl (HBP), which accumulates in the medium. This phenotype is due to the expression of the plasmid-encoded DBT-desulphurization (dsz) operon, which encodes three proteins, DszA, B and C. In this paper it is shown, using [35S]DBT radiolabelling studies, that sulphur is released in the form of inorganic sulphite. The pathway of DBT desulphurization is described in detail. In summary, DszC catalyses the stepwise S-oxidation of DBT, first to dibenzothiophene 5-oxide (DBTO) and then to dibenzothiophene 5,5-dioxide (DBTO2); DszA catalyses the conversion of DBTO2 to 2-(2'-hydroxyphenyl)benzene sulphinate (HBPSi-) and DszB catalyses the desulphination of HBPSi- to give HBP and sulphite. Studies with cell-free extracts show that DszA and DszC, but not DszB, require NADH for activity. 18O2-labelling studies show that each incorporated oxygen atom is derived directly from molecular oxygen. These results are consistent with the role of DszC as a mono-oxygenase, of DszA as an apparently unique enzyme which catalyses the reductive hydroxylation of DBTO2 leading to cleavage of the thiophene ring, and of DszB as an aromatic sulphinic acid hydrolase.

  2. Identification and characterization of the afsR homologue regulatory gene from Streptomyces peucetius ATCC 27952.

    PubMed

    Parajuli, Niranjan; Viet, Hung Trinh; Ishida, Kenji; Tong, Hang Thi; Lee, Hei Chan; Liou, Kwangkyoung; Sohng, Jae Kyung

    2005-01-01

    We have isolated an afsR homologue, called afsR-p, through genome analysis of Streptomyces peucetius ATCC 27952. AfsR-p shares 60% sequence identity with AfsR from Streptomyces coelicolor A3 (2). afsR-p was expressed under the control of the ermE* promoter in its hosts S. peucetius, Streptomyces lividans TK 24, Streptomyces clavuligerus and Streptomyces griseus. We observed overproduction of doxorubicin (4-fold) in S. peucetius, gamma-actinorhodin (2.6-fold) in S. lividans, clavulanic acid (1.5-fold) in S. clavuligerus and streptomycin (slight) in S. griseus. Overproduction was due to expression of the gene in these strains as compared to the wild-type strains harboring the vector only. Comparative study of the expression of afsR-p revealed that regulatory networking in Streptomyces is not uniform. We speculate that phosphorylated AfsR-p becomes bound to the promoter region of afsS. The latter activates other regulatory genes, including pathway regulatory genes, and induces the production of secondary metabolites including antibiotics. We identified specific conserved amino acids and exploited them for the isolation of the partial sequence of the afsR homologue from S. clavuligerus and Streptomyces achromogens (rubradirin producer). Such findings provide additional evidence for the presence of a serine/threonine and tyrosine kinase-dependent global regulatory network in Streptomyces. PMID:15921897

  3. Biodesulfurization of a system containing synthetic fuel using Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, Danielle; de Oliveira, Débora; de Souza, Antônio A Ulson; Souza, Selene M A Guelli U

    2014-11-01

    The burning of fossil fuels has released a large quantity of pollutants into the atmosphere. In this context, sulfur dioxide is one of the most noxious gas which, on reacting with moist air, is transformed into sulfuric acid, causing the acid rain. In response, many countries have reformulated their legislation in order to enforce the commercialization of fuels with very low sulfur levels. The existing desulfurization processes cannot remove such low levels of sulfur and thus a biodesulfurization has been developed, where the degradation of sulfur occurs through the action of microorganisms. Rhodococcus erythropolis has been identified as one of the most promising bacteria for use in the biodesulfurization. In this study, the effectiveness of the strain R. erythropolis ATCC 4277 in the desulfurization of dibenzothiophene (DBT) was evaluated in a batch reactor using an organic phase (n-dodecane or diesel) concentrations of 20, 80, and 100 % (v/v). This strain was able to degrade 93.3, 98.0, and 95.5 % of the DBT in the presence of 20, 80, and 100 % (v/v) of dodecane, respectively. The highest value for the specific DBT degradation rate was 44 mmol DBT · kg DCW(-1) · h(-1), attained in the reactor containing 80 % (v/v) of n-dodecane as the organic phase. PMID:25163887

  4. Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975.

    PubMed Central

    Song, D D; Jacques, N A

    1999-01-01

    The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 degrees C, indicating that Asp-312 present in the 'sucrose box' was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a beta-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius. PMID:10548559

  5. Compositional and toxicological evaluation of the diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142

    NASA Technical Reports Server (NTRS)

    Schneegurt, M. A.; Arieli, B.; McKeehen, J. D.; Stephens, S. D.; Nielsen, S. S.; Saha, P. R.; Trumbo, P. R.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

    1995-01-01

    Compositional analyses of Cyanothece sp. strain ATCC 51142 showed high protein (50-60%) and low fat (0.4-1%) content, and the ability to synthesize vitamin B12. The amino acid profile indicated that Cyanothece sp. was a balanced protein source. Fatty acids of the 18:3n-3 type were also present. Mineral analyses indicated that the cellular biomass may be a good source of Fe, Zn and Na. Caloric content was 4.5 to 5.1 kcal g dry weight-1 and the carbon content was approximately 40% on a dry weight basis. Nitrogen content was 8 to 9% on a dry weight basis and total nucleic acids were 1.3% on a dry weight basis. Short-term feeding studies in rats followed by histopathology found no toxicity or dietary incompatibility problems. The level of uric acid and allantoin in urine and tissues was low, suggesting no excess of nucleic acids, as sometimes reported in the past for a cyanobacteria-containing diet. The current work discusses the potential implications of these results for human nutrition applications.

  6. Sophorolipids Production by Candida bombicola ATCC 22214 and its Potential Application in Microbial Enhanced Oil Recovery.

    PubMed

    Elshafie, Abdulkadir E; Joshi, Sanket J; Al-Wahaibi, Yahya M; Al-Bemani, Ali S; Al-Bahry, Saif N; Al-Maqbali, Dua'a; Banat, Ibrahim M

    2015-01-01

    Biosurfactant production using Candida bombicola ATCC 22214, its characterization and potential applications in enhancing oil recovery were studied at laboratory scale. The seed media and the production media were standardized for optimal growth and biosurfactant production. The production media were tested with different carbon sources: glucose (2%w/v) and corn oil (10%v/v) added separately or concurrently. The samples were collected at 24 h interval up to 120 h and checked for growth (OD660), and biosurfactant production [surface tension (ST) and interfacial tension (IFT)]. The medium with both glucose and corn oil gave better biosurfactant production and reduced both ST and IFT to 28.56 + 0.42mN/m and 2.13 + 0.09mN/m, respectively within 72 h. The produced biosurfactant was quite stable at 13-15% salinity, pH range of 2-12, and at temperature up to 100°C. It also produced stable emulsions (%E24) with different hydrocarbons (pentane, hexane, heptane, tridecane, tetradecane, hexadecane, 1-methylnaphthalene, 2,2,4,4,6,8-heptamethylnonane, light and heavy crude oil). The produced biosurfactant was extracted using ethyl acetate and characterized as a mixture of sophorolipids (SPLs). The potential of SPLs in enhancing oil recovery was tested using core-flooding experiments under reservoir conditions, where additional 27.27% of residual oil (Sor) was recovered. This confirmed the potential of SPLs for applications in microbial enhanced oil recovery. PMID:26635782

  7. Legionella oakridgensis ATCC 33761 genome sequence and phenotypic characterization reveals its replication capacity in amoebae.

    PubMed

    Brzuszkiewicz, Elzbieta; Schulz, Tino; Rydzewski, Kerstin; Daniel, Rolf; Gillmaier, Nadine; Dittmann, Christine; Holland, Gudrun; Schunder, Eva; Lautner, Monika; Eisenreich, Wolfgang; Lück, Christian; Heuner, Klaus

    2013-12-01

    Legionella oakridgensis is able to cause Legionnaires' disease, but is less virulent compared to L. pneumophila strains and very rarely associated with human disease. L. oakridgensis is the only species of the family legionellae which is able to grow on media without additional cysteine. In contrast to earlier publications, we found that L. oakridgensis is able to multiply in amoebae. We sequenced the genome of L. oakridgensis type strain OR-10 (ATCC 33761). The genome is smaller than the other yet sequenced Legionella genomes and has a higher G+C-content of 40.9%. L. oakridgensis lacks a flagellum and it also lacks all genes of the flagellar regulon except of the alternative sigma-28 factor FliA and the anti-sigma-28 factor FlgM. Genes encoding structural components of type I, type II, type IV Lvh and type IV Dot/Icm, Sec- and Tat-secretion systems could be identified. Only a limited set of Dot/Icm effector proteins have been recognized within the genome sequence of L. oakridgensis. Like in L. pneumophila strains, various proteins with eukaryotic motifs and eukaryote-like proteins were detected. We could demonstrate that the Dot/Icm system is essential for intracellular replication of L. oakridgensis. Furthermore, we identified new putative virulence factors of Legionella.

  8. Construction and evaluation of a Clostridium thermocellum ATCC 27405 whole-genome oligonucleotide microarray

    SciTech Connect

    Brown, Steven David; Raman, Babu; McKeown, Catherine K; Kale, Shubhangi P; He, Zhili; Mielenz, Jonathan R

    2007-04-01

    Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.

  9. Metabolic engineering of Corynebacterium glutamicum strain ATCC13032 to produce L-methionine.

    PubMed

    Qin, Tianyu; Hu, Xiaoqing; Hu, Jinyu; Wang, Xiaoyuan

    2015-01-01

    L-Methionine-producing strain QW102/pJYW-4-hom(m) -lysC(m) -brnFE was developed from Corynebacterium glutamicum strain ATCC13032, using metabolic engineering strategies. These strategies involved (i) deletion of the gene thrB encoding homoserine kinase to increase the precursor supply, (ii) deletion of the gene mcbR encoding the regulator McbR to release the transcriptional repression to various genes in the l-methionine biosynthetic pathway, (iii) overexpression of the gene lysC(m) encoding feedback-resistant aspartate kinase and the gene hom(m) encoding feedback-resistant homoserine dehydrogenase to further increase the precursor supply, and (iv) overexpression of the gene cluster brnF and brnE encoding the export protein complex BrnFE to increase extracellular l-methionine concentration. QW102/pJYW-4-hom(m) -lysC(m) -brnFE produced 42.2 mM (6.3 g/L) l-methionine after 64-H fed-batch fermentation. These results suggest that l-methionine-producing strains can be developed from wild-type C. glutamicum strains by rationally metabolic engineering.

  10. Pivotal role of anthranilate dioxygenase genes in the adaptation of Burkholderia multivorans ATCC 17616 in soil.

    PubMed

    Nishiyama, Eri; Ohtsubo, Yoshiyuki; Yamamoto, Yasuhiro; Nagata, Yuji; Tsuda, Masataka

    2012-05-01

    In our recent screen for soil-induced genes, the expression of andA operon (andAcAdAbAa) for anthranilate catabolism in Burkholderia multivorans ATCC 17616 was found to increase dramatically in a soil sample (Nishiyama et al., Environ Microbiol 12: 2539, 2010). The operon was preceded by andR encoding a putative transcriptional regulator for the andA operon. In this study, the andA promoter was induced by tryptophan and anthranilate in an andR-dependent manner. The andA promoter in a deletion mutant lacking tryptophan dioxygenase (one of enzymes for the catabolism of tryptophan to anthranilate) did not respond to tryptophan, indicating that not tryptophan but anthranilate is the effector of AndR. Although both anthranilate and tryptophan were under the detection levels in the soil sample, andA promoter showed higher activity in the soil sample than in a laboratory medium. Such induction required andR and was moderately dependent on the ferric uptake regulator (Fur). The proliferation ability of andAc mutant in the sterile soil was low compared with the co-incubated wild-type cells. These findings suggested that in the soil environment, anthranilate dioxygenase genes are induced by AndR and Fur, and play a pivotal role in the proliferation in the soil environment. PMID:22360670

  11. Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032

    SciTech Connect

    Xu Ying; Yan Dazhong; Zhou Ningyi . E-mail: n.zhou@pentium.whiov.ac.cn

    2006-07-28

    Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.

  12. Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824

    PubMed Central

    Lee, Song F.; Forsberg, Cecil W.; Rattray, James B.

    1987-01-01

    Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-β-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar Km and Vmax values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity. Images PMID:16347312

  13. Biosurfactant-mediated biodegradation of straight and methyl-branched alkanes by Pseudomonas aeruginosa ATCC 55925

    PubMed Central

    2011-01-01

    Accidental oil spills and waste disposal are important sources for environmental pollution. We investigated the biodegradation of alkanes by Pseudomonas aeruginosa ATCC 55925 in relation to a rhamnolipid surfactant produced by the same bacterial strain. Results showed that the linear C11-C21 compounds in a heating oil sample degraded from 6% to 100%, whereas the iso-alkanes tended to be recalcitrant unless they were exposed to the biosurfactant; under such condition total biodegradation was achieved. Only the biodegradation of the commercial C12-C19 alkanes could be demonstrated, ranging from 23% to 100%, depending on the experimental conditions. Pristane (a C19 branched alkane) only biodegraded when present alone with the biosurfactant and when included in an artificial mixture even without the biosurfactant. In all cases the biosurfactant significantly enhanced biodegradation. The electron scanning microscopy showed that cells depicted several adaptations to growth on hydrocarbons, such as biopolymeric spheres with embedded cells distributed over different layers on the spherical surfaces and cells linked to each other by extracellular appendages. Electron transmission microscopy revealed transparent inclusions, which were associated with hydrocarbon based-culture cells. These patterns of hydrocarbon biodegradation and cell adaptations depended on the substrate bioavailability, type and length of hydrocarbon. PMID:21906343

  14. Production of Surfactant from Bacillus subtilis ATCC 21332 using Potato substrates

    SciTech Connect

    Fox, Sandra Lynn; Bala, Greg Alan

    2000-12-01

    Surfactin, a lipopeptide biosurfactant, produced by Bacillus subtilis is known to reduce the surface tension of water from 72 to 27 mN/m. Potato substrates were evaluated as a carbon source for surfactant production by B. subtilis ATCC 21332. An established potato medium, simulated liquid and solid potato waste media, and a commercially prepared potato starch in a mineral salts medium were evaluated in shake flask experiments to verify growth, surface tension reduction, and carbohydrate reduction capabilities. Total carbohydrate assays and glucose monitoring indicated that B. subtilis was able to degrade potato substrates to produce surfactant. Surface tensions dropped from 71.3±0.1 to 28.3±0.3 mN/m (simulated solid potato medium) and to 27.5±0.3 mN/m (mineral salts medium). A critical micelle concentration (CMC) of 0.10 g/l was obtained from a methylene chloride extract of the simulated solid potato medium.

  15. Statistical approach to optimization of fermentative production of gellan gum from Sphingomonas paucimobilis ATCC 31461.

    PubMed

    Bajaj, Ishwar B; Saudagar, Parag S; Singhal, Rekha S; Pandey, Ashok

    2006-09-01

    Gellan gum, a high-molecular-weight anionic linear polysaccharide produced by pure-culture fermentation from Sphingomonas paucimobilis ATCC 31461, has elicited industrial interest in recent years as a high-viscosity biogum, a suspending agent, a gelling agent, and an agar substitute in microbial media. In this paper we report on the optimization of gellan gum production using a statistical approach. In the first step, the one factor-at-a-time method was used to investigate the effect of medium constituents such as carbon and nitrogen sources; subsequently, the intuitive analysis based on statistical calculations carried out using the L16 -orthogonal array method. The design for the L16 -orthogonal array was developed and analyzed using MINITAB 13.30 software. All the fermentation runs were carried out at 30+/-2 degrees C on a rotary orbital shaker at 180 rpm for 48 h. In the second step, the effects of amino acids and gellan precursors such as uridine-5'-diphospate (UDP) and adenosine-5'-diphospate (ADP) on the fermentative production of gellan gum were studied. Media containing 4% soluble starch, 0.025% yeast extract, 1.0 mM ADP and 0.05% tryptophan gave a maximum yield of 43.6 g l(-1) starch-free gellan gum, which was significantly higher than reported values in the literature.

  16. Modeling for gellan gum production by Sphingomonas paucimobilis ATCC 31461 in a simplified medium.

    PubMed

    Wang, Xia; Xu, Ping; Yuan, Yong; Liu, Changlong; Zhang, Dezhong; Yang, Zhengting; Yang, Chunyu; Ma, Cuiqing

    2006-05-01

    Gellan gum production was carried out by Sphingomonas paucimobilis ATCC 31461 in a simplified medium with a short incubation time, and a kinetic model for understanding, controlling, and optimizing the fermentation process was proposed. The results revealed that glucose was the best carbon source and that the optimal concentration was 30 g liter(-1). As for the fermenting parameters, considerably large amounts of gellan gum were yielded by an 8-h-old culture and a 4% inoculum at 200 rpm on a rotary shaker. Under the optimized conditions, the maximum level of gellan gum (14.75 g liter(-1)) and the highest conversion efficiency (49.17%) were obtained in a 30-liter fermentor in batch fermentation. Logistic and Luedeking-Piret models were confirmed to provide a good description of gellan gum fermentation, which gave some support for the study of gellan gum fermentation kinetics. Additionally, this study is the first demonstration that gellan gum production is largely growth associated by analysis of kinetics in its batch fermentation process. Based on model prediction, higher gellan gum production (17.71 g liter(-1)) and higher conversion efficiency (57.12%) were obtained in fed-batch fermentation at the same total glucose concentration (30 g liter(-1)).

  17. Glucose metabolism in the antibiotic producing actinomycete Nonomuraea sp. ATCC 39727.

    PubMed

    Gunnarsson, Nina; Bruheim, Per; Nielsen, Jens

    2004-12-01

    The actinomycete Nonomuraea sp. ATCC 39727, producer of the glycopeptide A40926 that is used as precursor for the novel antibiotic dalbavancin, has an unusual carbon metabolism. Glucose is primarily metabolized via the Entner-Doudoroff (ED) pathway, although the energetically more favorable Embden-Meyerhof-Parnas (EMP) pathway is present in this organism. Moreover, Nonomuraea utilizes a PPi-dependent phosphofructokinase, an enzyme that has been connected with anaerobic metabolism in eukaryotes and higher plants, but recently has been recognized in several actinomycetes. In order to study its primary carbon metabolism in further detail, Nonomuraea was cultivated with [1-13C] glucose as the only carbon source and the 13C-labeling patterns of proteinogenic amino acids were determined by GC-MS analysis. Through this method, the fluxes in the central carbon metabolism during balanced growth were estimated. Moreover, a shift in the label incorporation pattern was observed in connection with phosphate limitation and increased antibiotic productivity in Nonomuraea. The shift indicated an increased flux through the EMP pathway at the expense of the flux through the ED pathway, a suggestion that was supported by alterations in intracellular metabolite levels during phosphate limitation. In contrast, expression levels of genes encoding enzymes in the ED and EMP pathways were not affected by phosphate limitation.

  18. Relationship between glycocalyx and povidone-iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms.

    PubMed

    Brown, M L; Aldrich, H C; Gauthier, J J

    1995-01-01

    Biofilm-embedded bacteria are generally more resistant to antimicrobial agents than are planktonic bacteria. Two possible mechanisms for biofilm resistance are that the glycocalyx matrix secreted by cells in a biofilm reacts with and neutralizes the antimicrobial agent and that the matrix creates a diffusion barrier to the antimicrobial agent. This study was therefore conducted to examine the relationship between glycocalyx and enhanced povidone-iodine resistance in biofilms of Pseudomonas aeruginosa (ATCC 27853). Biofilms were generated by inoculation of polycarbonate membranes with broth-grown cells and incubation of them on the surfaces of nutrient agar plates. The quantities of glycocalyx material per cell were found not to be significantly different between biofilm and planktonic samples. Transmission electron microscopy showed that the distributions of glycocalyx material around cells differed in biofilm and in planktonic samples. Addition of alginic acid to planktonic cell suspensions resulted in a slight increase in resistance to povidone-iodine, suggesting some neutralizing interaction. However, the iodine demands created by biofilm and planktonic samples of equivalent biomass were not significantly different and, therefore, do not explain the contrast in resistance observed between biofilm and planktonic samples. Examination of the relationship between cell death and biomass detachment from the glycocalyx matrix revealed that most cell death occurred in the fraction of biomass that detached from a biofilm during treatment. The overall rate of iodine diffusion through biofilms was not different from that of planktonic cells collected on a polycarbonate membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. In vitro and in vivo activities of ticarcillin-loaded nanoliposomes with different surface charges against Pseudomonas aeruginosa (ATCC 29248)

    PubMed Central

    2012-01-01

    Background Pseudomonas aeruginosa exhibits multiple antibiotic resistance mechanisms. Different studies have shown that entrapment of antibiotics into liposomes could increase their anti-Pseudomonas activity. The objectives of this study were to prepare ticarcillin loaded-nanoliposomes with variable surface charges and evaluate their in vitro and in vivo efficacies against Pseudomonas aeruginosa (ATCC 29248). Methods Ticarcillin-loaded nanoliposomes with positive, negative and neutral surface charges were prepared by extrusion method. Ticarcillin encapsulation efficacies for different formulations were measured by HPLC method. Minimum inhibitory concentration (MIC) of ticarcillin nanoliposomal forms against strain ATCC 29248 were determined by broth dilution method. The killing rate of Pseudomonas aeruginosa was exposed to various concentrations of ticarcillin in free and nanoliposomal forms were analyzed. Ultimately, in vivo therapeutic efficacy of nanoliposomes in burned mice skin infected with strain ATCC 29248 was investigated. Results The encapsulation efficacies for ticarcillin-loaded cationic nanoliposomes were significantly higher (76% ± 0.17) than those of neutral (55% ± 0.14) and anionic (43% ± 0.14) nanoliposomes. The MIC of free, cationic, neutral and anionic nanoliposomal forms of ticarcillin against ATCC 29248 were to 24, 3, 6 and 48 mg/L, respectively. The killing rates of ticarcillin-loaded cationic nanoliposomes were higher than those of free and other drug formulations. Treatment by ticarcillin-loaded nanoliposomes with positive, neutral and negative surface charges resulted in almost 100, 60 and 20% survival rates, respectively. Conclusion Our data suggested that cationic ticarcillin-loaded nanoliposomes because of high effectiveness would be a good choice to treatment of Pseudomonas aeruginosa infections. PMID:23351156

  20. Co-fermentation of carbon sources by Enterobacter aerogenes ATCC 29007 to enhance the production of bioethanol.

    PubMed

    Thapa, Laxmi Prasad; Lee, Sang Jun; Yang, Xiao Guang; Yoo, Hah Young; Kim, Sung Bong; Park, Chulhwan; Kim, Seung Wook

    2014-06-01

    We investigated the enhancement of bioethanol production in Enterobacter aerogenes ATCC 29007 by co-fermentation of carbon sources such as glycerol, glucose, galactose, sucrose, fructose, xylose, starch, mannitol and citric acid. Biofuel production increases with increasing growth rate of microorganisms; that is why we investigated the optimal growth rate of E. aerogenes ATCC 29007, using mixtures of different carbon sources with glycerol. E. aerogenes ATCC 29007 was incubated in media containing each carbon source and glycerol; growth rate and bioethanol production improved in all cases compared to those in medium containing glycerol alone. The growth rate and bioethanol production were highest with mannitol. Fermentation was carried out at 37 °C for 18 h, pH 7, using 50 mL defined production medium in 100 mL serum bottles at 200 rpm. Bioethanol production under optimized conditions in medium containing 16 g/L mannitol and 20 g/L glycerol increased sixfold (32.10 g/L) than that containing glycerol alone (5.23 g/L) as the carbon source in anaerobic conditions. Similarly, bioethanol production using free cells in continuous co-fermentation also improved (27.28 g/L) when 90.37 % of 16 g/L mannitol and 67.15 % of 20 g/L glycerol were used. Although naturally existing or engineered microorganisms can ferment mixed sugars sequentially, the preferential utilization of glucose to non-glucose sugars often results in lower overall yield and productivity of ethanol. Here, we present new findings in E. aerogenes ATCC 29007 that can be used to improve bioethanol production by simultaneous co-fermentation of glycerol and mannitol.

  1. Transcription profiling of interactions between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 during Cheddar cheese simulation.

    PubMed

    Desfossés-Foucault, Émilie; LaPointe, Gisèle; Roy, Denis

    2014-05-16

    The starter cultures (Lactococcus sp.) and non-starter lactic acid bacteria (mostly Lactobacillus spp.) are essential to flavor development of Cheddar cheese. The aim of this study was to elucidate the transcriptional interaction between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 in mixed cultures during simulated Cheddar cheese manufacture (Pearce activity test) and ripening (slurry). Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of 34 genes common to both bacteria and for eight genes specific to either L. lactis subsp. cremoris SK11 or L. paracasei ATCC 334. The multifactorial analysis (MFA) performed on fold change results for each gene revealed that the genes linked to stress, protein and peptide degradation as well as carbohydrate metabolism of L. paracasei ATCC 334 were especially overexpressed in mixed culture with L. lactis subsp. cremoris SK11 during the ripening simulation. For L. lactis subsp. cremoris SK11, genes coding for amino acid metabolism were more expressed during the cheese manufacture simulation, especially in single culture. These results show how complementary functions of starter and NSLAB contribute to activities useful for flavor development. PMID:24674930

  2. Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

    PubMed

    Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun; Ryu, Sangryeol

    2015-01-01

    The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage.

  3. Transcriptome analysis of Cronobacter sakazakii ATCC BAA-894 after interaction with human intestinal epithelial cell line HCT-8.

    PubMed

    Jing, Chun-e; Du, Xin-jun; Li, Ping; Wang, Shuo

    2016-01-01

    Cronobacter spp. are opportunistic pathogens that are responsible for infections including severe meningitis, septicemia, and necrotizing enterocolitis in neonates and infants. To date, questions still remain regarding the mechanisms of pathogenicity and virulence determinants for each bacterial strain. In this study, we established an in vitro model for Cronobacter sakazakii ATCC BAA-894 infection of HCT-8 human colorectal epithelial cells. The transcriptome profile of C. sakazakii ATCC BAA-894 after interaction with HCT-8 cells was determined using high-throughput whole-transcriptome sequencing (RNA sequencing (RNA-seq)). Gene expression profiles indicated that 139 genes were upregulated and 72 genes were downregulated in the adherent C. sakazakii ATCC BAA-894 strain on HCT-8 cells compared to the cultured bacteria in the cell-free medium. Expressions of some flagella genes and virulence factors involved in adherence were upregulated. High osmolarity and osmotic stress-associated genes were highly upregulated, as well as genes responsible for the synthesis of lipopolysaccharides and outer membrane proteins, iron acquisition systems, and glycerol and glycerophospholipid metabolism. In sum, our study provides further insight into the mechanisms underlying C. sakazakii pathogenesis in the human gastrointestinal tract. PMID:26481623

  4. Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella.

    PubMed

    Vilela, Simone F G; Barbosa, Júnia O; Rossoni, Rodnei D; Santos, Jéssica D; Prata, Marcia C A; Anbinder, Ana Lia; Jorge, Antonio O C; Junqueira, Juliana C

    2015-01-01

    Probiotic strains of Lactobacillus have been studied for their inhibitory effects on Candida albicans. However, few studies have investigated the effect of these strains on biofilm formation, filamentation and C. albicans infection. The objective of this study was to evaluate the influence of Lactobacillus acidophilus ATCC 4356 on C. albicans ATCC 18804 using in vitro and in vivo models. In vitro analysis evaluated the effects of L. acidophilus on the biofilm formation and on the capacity of C. albicans filamentation. For in vivo study, Galleria mellonella was used as an infection model to evaluate the effects of L. acidophilus on candidiasis by survival analysis, quantification of C. albicans CFU/mL, and histological analysis. The direct effects of L. acidophilus cells on C. albicans, as well as the indirect effects using only a Lactobacillus culture filtrate, were evaluated in both tests. The in vitro results showed that both L. acidophilus cells and filtrate were able to inhibit C. albicans biofilm formation and filamentation. In the in vivo study, injection of L. acidophilus into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, the number of C. albicans CFU/mL recovered from the larval hemolymph was lower in the group inoculated with L. acidophilus compared to the control group. In conclusion, L. acidophilus ATCC 4356 inhibited in vitro biofilm formation by C. albicans and protected G. mellonella against experimental candidiasis in vivo.

  5. Microencapsulated Bifidobacterium longum subsp. infantis ATCC 15697 favorably modulates gut microbiota and reduces circulating endotoxins in F344 rats.

    PubMed

    Rodes, Laetitia; Saha, Shyamali; Tomaro-Duchesneau, Catherine; Prakash, Satya

    2014-01-01

    The gut microbiota is a bacterial bioreactor whose composition is an asset for human health. However, circulating gut microbiota derived endotoxins cause metabolic endotoxemia, promoting metabolic and liver diseases. This study investigates the potential of orally delivered microencapsulated Bifidobacterium infantis ATCC 15697 to modulate the gut microbiota and reduce endotoxemia in F344 rats. The rats were gavaged daily with saline or microencapsulated B. infantis ATCC 15697. Following 38 days of supplementation, the treated rats showed a significant (P < 0.05) increase in fecal Bifidobacteria (4.34 ± 0.46 versus 2.45 ± 0.25% of total) and B. infantis (0.28 ± 0.21 versus 0.52 ± 0.12 % of total) and a significant (P < 0.05) decrease in fecal Enterobacteriaceae (0.80 ± 0.45 versus 2.83 ± 0.63% of total) compared to the saline control. In addition, supplementation with the probiotic formulation reduced fecal (10.52 ± 0.18 versus 11.29 ± 0.16 EU/mg; P = 0.01) and serum (0.33 ± 0.015 versus 0.30 ± 0.015 EU/mL; P = 0.25) endotoxins. Thus, microencapsulated B. infantis ATCC 15697 modulates the gut microbiota and reduces colonic and serum endotoxins. Future preclinical studies should investigate the potential of the novel probiotic formulation in metabolic and liver diseases. PMID:24967382

  6. Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli

    PubMed Central

    Ma, Tian; Zhou, Yuanjie; Li, Xiaowei; Zhu, Fayin; Cheng, Yongbo; Liu, Yi; Deng, Zixin

    2015-01-01

    Abstract As a highly valued keto‐carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α‐Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole‐genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio‐Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high‐efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4‐fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future. PMID:26580858

  7. Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella

    PubMed Central

    Vilela, Simone FG; Barbosa, Júnia O; Rossoni, Rodnei D; Santos, Jéssica D; Prata, Marcia CA; Anbinder, Ana Lia; Jorge, Antonio OC; Junqueira, Juliana C

    2015-01-01

    Probiotic strains of Lactobacillus have been studied for their inhibitory effects on Candida albicans. However, few studies have investigated the effect of these strains on biofilm formation, filamentation and C. albicans infection. The objective of this study was to evaluate the influence of Lactobacillus acidophilus ATCC 4356 on C. albicans ATCC 18804 using in vitro and in vivo models. In vitro analysis evaluated the effects of L. acidophilus on the biofilm formation and on the capacity of C. albicans filamentation. For in vivo study, Galleria mellonella was used as an infection model to evaluate the effects of L. acidophilus on candidiasis by survival analysis, quantification of C. albicans CFU/mL, and histological analysis. The direct effects of L. acidophilus cells on C. albicans, as well as the indirect effects using only a Lactobacillus culture filtrate, were evaluated in both tests. The in vitro results showed that both L. acidophilus cells and filtrate were able to inhibit C. albicans biofilm formation and filamentation. In the in vivo study, injection of L. acidophilus into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, the number of C. albicans CFU/mL recovered from the larval hemolymph was lower in the group inoculated with L. acidophilus compared to the control group. In conclusion, L. acidophilus ATCC 4356 inhibited in vitro biofilm formation by C. albicans and protected G. mellonella against experimental candidiasis in vivo. PMID:25654408

  8. Selection of the Strain Lactobacillus acidophilus ATCC 43121 and Its Application to Brewers' Spent Grain Conversion into Lactic Acid

    PubMed Central

    Liguori, Rossana; Soccol, Carlos Ricardo; Vandenberghe, Luciana Porto de Souza; Woiciechowski, Adenise Lorenci; Ionata, Elena; Marcolongo, Loredana; Faraco, Vincenza

    2015-01-01

    Six Lactobacillus strains were analyzed to select a bacterium for conversion of brewers' spent grain (BSG) into lactic acid. Among the investigated strains, L. acidophilus ATCC 43121 showed the highest yield of lactic acid production (16.1 g/L after 48 hours) when grown in a synthetic medium. It was then analyzed for its ability to grow on the hydrolysates obtained from BSG after acid-alkaline (AAT) or aqueous ammonia soaking (AAS) pretreatment. The lactic acid production by L. acidophilus ATCC 43121 through fermentation of the hydrolysate from AAS treated BSG was 96% higher than that from the AAT treated one, although similar yields of lactic acid per consumed glucose were achieved due to a higher (46%) glucose consumption by L. acidophilus ATCC 43121 in the AAS BSG hydrolysate. It is worth noting that adding yeast extract to the BSG hydrolysates increased both the yield of lactic acid per substrate consumed and the volumetric productivity. The best results were obtained by fermentation of AAS BSG hydrolysate supplemented by yeast extract, in which the strain produced 22.16 g/L of lactic acid (yield of 0.61 g/g), 27% higher than the value (17.49 g/L) obtained in the absence of a nitrogen source. PMID:26640784

  9. Selection of the Strain Lactobacillus acidophilus ATCC 43121 and Its Application to Brewers' Spent Grain Conversion into Lactic Acid.

    PubMed

    Liguori, Rossana; Soccol, Carlos Ricardo; Vandenberghe, Luciana Porto de Souza; Woiciechowski, Adenise Lorenci; Ionata, Elena; Marcolongo, Loredana; Faraco, Vincenza

    2015-01-01

    Six Lactobacillus strains were analyzed to select a bacterium for conversion of brewers' spent grain (BSG) into lactic acid. Among the investigated strains, L. acidophilus ATCC 43121 showed the highest yield of lactic acid production (16.1 g/L after 48 hours) when grown in a synthetic medium. It was then analyzed for its ability to grow on the hydrolysates obtained from BSG after acid-alkaline (AAT) or aqueous ammonia soaking (AAS) pretreatment. The lactic acid production by L. acidophilus ATCC 43121 through fermentation of the hydrolysate from AAS treated BSG was 96% higher than that from the AAT treated one, although similar yields of lactic acid per consumed glucose were achieved due to a higher (46%) glucose consumption by L. acidophilus ATCC 43121 in the AAS BSG hydrolysate. It is worth noting that adding yeast extract to the BSG hydrolysates increased both the yield of lactic acid per substrate consumed and the volumetric productivity. The best results were obtained by fermentation of AAS BSG hydrolysate supplemented by yeast extract, in which the strain produced 22.16 g/L of lactic acid (yield of 0.61 g/g), 27% higher than the value (17.49 g/L) obtained in the absence of a nitrogen source.

  10. Sensitive and specific modified Hodge test for KPC and metallo-beta- lactamase detection in Pseudomonas aeruginosa by use of a novel indicator strain, Klebsiella pneumoniae ATCC 700603.

    PubMed

    Pasteran, Fernando; Veliz, Omar; Rapoport, Melina; Guerriero, Leonor; Corso, Alejandra

    2011-12-01

    We evaluated the ability of the modified Hodge test to discriminate between KPC- and metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa isolates and carbapenemase nonproducers. With Escherichia coli ATCC 25922 as the indicator strain, the MHT resulted in low sensitivity, specificity, and repeatability. Replacing the indicator strain with Klebsiella pneumoniae ATCC 700603 led to an improved performance (100%, 97%, 0%, and 100% sensitivity, specificity, indeterminate results and repeatability, respectively).

  11. Genome Sequences of Ralstonia insidiosa Type Strain ATCC 49129 and Strain FC1138, a Strong Biofilm Producer Isolated from a Fresh-Cut Produce-Processing Plant

    PubMed Central

    Xu, Yunfeng; Nagy, Attila; Yan, Xianghe; Haley, Bradd J.; Kim, Seon Woo; Liu, Nancy T.

    2016-01-01

    Ralstonia insidiosa is an opportunistic pathogen and a strong biofilm producer. Here, we present the complete genome sequences of R. insidiosa FC1138 and ATCC 49129. Both strains have two circular chromosomes of approximately 3.9 and 1.9 Mb and a 50-kb plasmid. ATCC 49129 also possesses a megaplasmid of approximately 318 kb. PMID:27540070

  12. Augmentation of Actinobacillus actinomycetemcomitans Invasion of Human Oral Epithelial Cells and Up-Regulation of Interleukin-8 Production by Saliva CD14

    PubMed Central

    Takayama, Atsuko; Satoh, Aya; Ngai, Tomoko; Nishimura, Takashi; Ikawa, Keiji; Matsuyama, Takami; Shimauchi, Hidetoshi; Takada, Haruhiko; Sugawara, Shunji

    2003-01-01

    It has recently been shown that human salivary glands constitutively express CD14, an important molecule in innate immunity, and that a soluble form of CD14 is secreted in saliva. The concentration of CD14 in parotid (a serous gland) saliva was comparable to that in normal serum and 10-fold the amount in whole saliva, although the physiological function of saliva CD14 remained unclear. Actinobacillus actinomycetemcomitans is a periodontopathic bacterium and is able to invade oral epithelial cells. The present study showed that upon exposure to live A. actinomycetemcomitans Y4 for 2 h, human oral epithelial HSC-2 cells produced interleukin-8 (IL-8) for a further 24 h and whole saliva augmented the production induced by A. actinomycetemcomitans Y4. Parotid saliva showed a more pronounced effect on the production of IL-8 than whole saliva. Neither saliva preparation itself had IL-8-inducing activity. Parotid saliva exhibited antibacterial activity against a low concentration of A. actinomycetemcomitans Y4, but recombinant CD14 did not show the activity. The internalization of A. actinomycetemcomitans Y4 into HSC-2 cells was inhibited by cytochalasin B, indicating that the process was actin dependent, and depletion of CD14 from parotid saliva inhibited the invasion and, as a consequence, inhibited production of IL-8. Furthermore, human recombinant CD14 augmented invasion and IL-8 production. These results suggest that saliva CD14 promoted the invasion of oral epithelial cells by A. actinomycetemcomitans and consequently augmented the production of IL-8, playing an important role in innate immunity in the oral cavity. PMID:14500479

  13. Indirect enzyme-linked immunosorbent assay for detection of antibody to a 110,000-molecular-weight hemolysin of Actinobacillus pleuropneumoniae.

    PubMed Central

    Ma, J N; Inzana, T J

    1990-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect swine antibody to a 110,000-molecular-weight hemolysin (110K hemolysin) of Actinobacillus pleuropneumoniae. Affinity-purified rabbit polyclonal or mouse monoclonal immunoglobulin G to the hemolysin of A. pleuropneumoniae serotype 5 strain J45, followed by hemolysin-rich concentrated culture supernatant, was used to bind swine antibody to hemolysin to microdilution plates. Sixty-nine serum samples from swine that were clinically normal, presented with clinical evidence of pleuropneumonia, were experimentally immunized or challenged, or were free of pleuropneumonia were tested, and their ELISA titers were compared with complement fixation (CF) titers. On the basis of serum samples from swine that were clinically normal and negative by CF, an ELISA titer of 1:320 or greater was considered positive. In comparison with CF, the sensitivity of the ELISA was 98.1% and the specificity was 90%. The two samples negative by CF and positive by indirect ELISA were, however, also positive for antibody to serotype 5 capsule by ELISA. Immunization of normal pigs with whole cells or purified hemolysin boosted titers 4- to 128-fold within 4 weeks. Immunoblotting demonstrated that the affinity-purified immunoglobulin G to hemolysin used for capture in the assay recognized only a 110K protein of A. pleuropneumoniae serotypes 1 to 7, although the reactivity was quantitatively variable between serotypes. Therefore, the indirect ELISA is capable of identifying animals infected with or exposed to most, if not all, serotypes of A. pleuropneumoniae. If an indirect ELISA titer of 1:320 or greater is considered positive, the assay can be a valuable diagnostic tool in both clinical and research laboratories. Images PMID:2380363

  14. Aerosol exposure of pigs to viable or inactivated Actinobacillus pleuropneumoniae serotype 9 induces antibodies in bronchoalveolar lining fluids and serum, and protects against homologous challenge.

    PubMed

    Hensel, A; Stockhofe-Zurwieden, N; Ganter, M; Petzoldt, K

    1995-11-01

    A dose-defined nose-only inhalation system for pigs was used to study the immunogenic and protective potentials of a single aerosol application of viable or killed Actinobacillus pleuropneumoniae serotype 9. Respiratory volumes were measured for each pig to calculate inhaled individual doses. Eight pigs inhaled 107 CFU A. pleuropneumoniae CVI 13261 reference strain for serotype 9. Another eight pigs received an identical dose of killed actinobacilli. After three weeks the pigs and nonexposed controls were challenged with 108 CFU of the homologous strain by aerosol. Bronchoalveolar lavage (BALF) in pigs was performed during the experiment to obtain lavage samples for assessment of local antibodies. Isotype-specific antibody responses in serum and BAL fluids were measured by ELISAs based on whole-cell antigens. The protective efficacy of aerosol immunization was evaluated by clinical and post-mortem examinations. The controls developed fever and severe pleuropneumonia, whereas previously exposed pigs had less fever and less extensive gross pulmonary lesions. After the first aerosol exposure pulmonary IgM, and IgG antibodies reactive with A. pleuropneumoniae increased significantly in both aerosol exposed groups. IgA in BALF and serum concentrations of each Ig class were significantly increased in the group exposed to viable bacteria when compared to the non-exposed controls. After aerosol challenge a pronounced increase of systemic and pulmonary IgA, IgM, and IgG antibodies was detected in both exposure groups. Aerosol application of whole-cell A. pleuropneumoniae bacterins induced similar protective effects against aerosol challenge infection as administration of an identical dose of viable bacteria. Inhalation of A. pleuropneumoniae may lead to asymptomatic carriers in some pigs that could spread the disease under field conditions.

  15. The Lon protease homologue LonA, not LonC, contributes to the stress tolerance and biofilm formation of Actinobacillus pleuropneumoniae.

    PubMed

    Xie, Fang; Li, Gang; Zhang, Yanhe; Zhou, Long; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

    2016-04-01

    Lon proteases are a family of ATP-dependent proteases that are involved in the degradation of abnormal proteins in bacteria exposed to adverse environmental stress. An analysis of the genome sequence of Actinobacillus pleuropneumoniae revealed the unusual presence of two putative ATP-dependent Lon homologues, LonA and LonC. Sequence comparisons indicated that LonA has the classical domain organization of the LonA subfamily, which includes the N-terminal domain, central ATPase (AAA) domain, and C-terminal proteolytic (P) domain. LonC belongs to the recently classified LonC subfamily, which includes Lon proteases that contain neither the N-terminal domain of LonA nor the transmembrane region that is present only in LonB subfamily members. To investigate the roles of LonA and LonC in A. pleuropneumoniae, mutants with deletions in the lonA and lonC genes were constructed. The impaired growth of the △lonA mutant exposed to low and high temperatures and osmotic and oxidative stress conditions indicates that the LonA protease is required for the stress tolerance of A. pleuropneumoniae. Furthermore, the △lonA mutant exhibited significantly reduced biofilm formation compared to the wild-type strain. However, no significant differences in stress responses or biofilm formation were observed between the △lonC mutant and the wild-type strain. The △lonA mutant exhibited reduced colonization ability and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model compared to the wild-type strain. Disruption of lonC gene did not significantly influence the colonization and virulence of A. pleuropneumoniae. The data presented in this study illustrate that the LonA protease, but not the LonC protease, is required for the stress tolerance, biofilm formation and pathogenicity of A. pleuropneumoniae.

  16. Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

    PubMed Central

    White, P A; Wilson, M; Nair, S P; Kirby, A C; Reddi, K; Henderson, B

    1995-01-01

    The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP. PMID:7790076

  17. Efficacy of tilmicosin phosphate (Pulmotil premix) in feed for the treatment of a clinical outbreak of Actinobacillus pleuropneumoniae infection in growing-finishing pigs.

    PubMed

    Hoflack, G; Maes, D; Mateusen, B; Verdonck, M; de Kruif, A

    2001-11-01

    A double-blind randomized clinical trial was carried out to investigate the efficacy of tilmicosin (Pulmotil premix) for the treatment of a clinical outbreak of Actinobacillus pleuropneumoniae infection in growing-finishing pigs. The effects of tilmicosin administration in the feed at 400 mg/kg and an injection therapy of clinically diseased pigs with long-acting oxytetracycline (Terramycine LA) at 20 mg/kg bodyweight were compared. Both groups, totalling 147 pigs, were compared during a medication period of 15 days and a post-medication period of 11 days by means of different clinical and performance parameters. During the medication period, the tilmicosin group showed a significant advantage with respect to the number of new disease cases (P < 0.01), and a non-significant advantage regarding the number of removed pigs (P = 0.16), the number of sick pigs that recovered (P = 0.27) and the time to recovery (P = 0.42). During the post-medication period, the pigs of the tilmicosin group showed numerical non-significant benefits (P > 0.05) with respect to the clinical parameters. During the overall study period (26 days), the average daily gain and the feed conversion ratio were both significantly (P < 0.01) better in pigs from the tilmicosin group compared with pigs from the oxytetracycline group. This study demonstrated that in-feed medication of tilmicosin at a dosage of 400 mg/kg is efficacious for the treatment of a clinical respiratory disease outbreak of A. pleuropneumoniae infection in growing-finishing pigs. Compared with oxytetracycline injection of clinically diseased pigs, the tilmicosin treatment is particularly beneficial in the prevention of new disease cases while increasing or maintaining the performance of the pigs.

  18. Activity of antibodies against Salmonella dublin, Toxoplasma gondii, or Actinobacillus pleuropneumoniae in sera after treatment with electron beam irradiation or binary ethylenimine.

    PubMed Central

    Kyvsgaard, N C; Lind, P; Preuss, T; Kamstrup, S; Lei, J C; Bøgh, H O; Nansen, P

    1996-01-01

    Viral contamination of biological material may constitute a risk when samples are exchanged between countries, and it may be necessary to subject the material to an inactivation treatment. The present study investigated possible adverse effects on antibody activity subsequent to either electron beam irradiation or binary ethylenimine (BEI) treatment. The treatments were performed with sera obtained from pigs or cattle. For each treatment level, the posttreatment activity was plotted against the pretreatment activity, and regression analyses were carried out. The slope of the regression line was used as an estimate for the relative posttreatment activity. For a Toxoplasma gondii indirect enzyme-linked immunosorbent assay (ELISA) and agglutination assay as well as for a Salmonella dublin indirect ELISA, the posttreatment activity was more than 89% of the pretreatment activity when the samples were irradiated in the frozen state (on dry ice) with up to 46. kGy or when they were treated with 5 or 10 mM BEI for up to 48 h. The samples were more sensitive to irradiation in the liquid state. Thus, samples irradiated with 22.6 kGy retained 98% of their activity in the indirect ELISA when they were irradiated in the frozen state on dry ice but only 35% of their activity when they were irradiated in the liquid state at 0 degrees C. The patterns seen in an S. dublin blocking ELISA and an Actinobacillus pleuropneumoniae complement fixation assay differed in that samples with a low level of pretreatment activity were subject to a relatively greater decrease in activity than samples with a high level of pretreatment activity. The complement fixation assay was particularly sensitive to irradiation of serum. It is concluded that serum samples retain sufficient activity by both methods of virus inactivation, especially when used in indirect ELISA or in the T. gondii agglutination assay. PMID:8914750

  19. Antibacterial activity of antagonistic bacterium Bacillus subtilis DJM-51 against phytopathogenic Clavibacter michiganense subsp. michiganense ATCC 7429 in vitro.

    PubMed

    Jung, W J; Mabood, F; Souleimanov, A; Whyte, L G; Niederberger, T D; Smith, D L

    2014-12-01

    To investigate antibacterial activity against the tomato pathogen Clavibacter michiganense subsp. michiganense ATCC 7429 (Cmm ATCC 7429), Bacillus subtilis DJM-51 was isolated from rhizosphere soil. For isolation of bacteria, samples were taken from rhizosphere soil. The isolate, DJA-51, had strong antagonistic ability against Tomato pathogen Cmm ATCC 7429 on nutrient-broth yeast extract agar (NBYA) as indicated by inhibition zones around colonies. On the basis of the nucleotide sequence of a conserved segment of the 16S rRNA gene, the bacterium has been identified as B. subtilis DJM-51. The growth of Cmm ATCC 7429 on NBYA plates was inhibited by culture broth of B. subtilis DJM-51 including cells, by the supernatant of culture broth of B. subtilis DJM-51, and by the liquid material resulting from butanol extract of bacterial cultures. The OD value in co-culture mixture was lower than the control throughout the entire incubation period. Antibiotics obtained from B. subtilis DJM-51 inhibited the growth of Tomato pathogen Cmm ATCC 7429. These results provide potentially information about the protection of tomato from pathogen Cmm ATCC 7429 under greenhouse conditions in Quebec.

  20. Antibacterial activity of antagonistic bacterium Bacillus subtilis DJM-51 against phytopathogenic Clavibacter michiganense subsp. michiganense ATCC 7429 in vitro.

    PubMed

    Jung, W J; Mabood, F; Souleimanov, A; Whyte, L G; Niederberger, T D; Smith, D L

    2014-12-01

    To investigate antibacterial activity against the tomato pathogen Clavibacter michiganense subsp. michiganense ATCC 7429 (Cmm ATCC 7429), Bacillus subtilis DJM-51 was isolated from rhizosphere soil. For isolation of bacteria, samples were taken from rhizosphere soil. The isolate, DJA-51, had strong antagonistic ability against Tomato pathogen Cmm ATCC 7429 on nutrient-broth yeast extract agar (NBYA) as indicated by inhibition zones around colonies. On the basis of the nucleotide sequence of a conserved segment of the 16S rRNA gene, the bacterium has been identified as B. subtilis DJM-51. The growth of Cmm ATCC 7429 on NBYA plates was inhibited by culture broth of B. subtilis DJM-51 including cells, by the supernatant of culture broth of B. subtilis DJM-51, and by the liquid material resulting from butanol extract of bacterial cultures. The OD value in co-culture mixture was lower than the control throughout the entire incubation period. Antibiotics obtained from B. subtilis DJM-51 inhibited the growth of Tomato pathogen Cmm ATCC 7429. These results provide potentially information about the protection of tomato from pathogen Cmm ATCC 7429 under greenhouse conditions in Quebec. PMID:25457795

  1. Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids

    SciTech Connect

    Wiesenborn, D.P.; Rudolph, F.B.; Papoutsakis, E.T. )

    1989-02-01

    Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the K{sub m} values at pH 7.5 and 30{degree}C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the K{sub m} value for acetoacetyl-CoA ranged from about 7 to 56{mu}M, depending on the acid substrate. The K{sub m} values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

  2. Mutational Studies of Putative Biosynthetic Genes for the Cyanobacterial Sunscreen Scytonemin in Nostoc punctiforme ATCC 29133

    PubMed Central

    Ferreira, Daniela; Garcia-Pichel, Ferran

    2016-01-01

    The heterocyclic indole-alkaloid scytonemin is a sunscreen found exclusively among cyanobacteria. An 18-gene cluster is responsible for scytonemin production in Nostoc punctiforme ATCC 29133. The upstream genes scyABCDEF in the cluster are proposed to be responsible for scytonemin biosynthesis from aromatic amino acid substrates. In vitro studies of ScyA, ScyB, and ScyC proved that these enzymes indeed catalyze initial pathway reactions. Here we characterize the role of ScyD, ScyE, and ScyF, which were logically predicted to be responsible for late biosynthetic steps, in the biological context of N. punctiforme. In-frame deletion mutants of each were constructed (ΔscyD, ΔscyE, and ΔscyF) and their phenotypes studied. Expectedly, ΔscyE presents a scytoneminless phenotype, but no accumulation of the predicted intermediaries. Surprisingly, ΔscyD retains scytonemin production, implying that it is not required for biosynthesis. Indeed, scyD presents an interesting evolutionary paradox: it likely originated in a duplication event from scyE, and unlike other genes in the operon, it has not been subjected to purifying selection. This would suggest that it is a pseudogene, and yet scyD is highly conserved in the scytonemin operon of cyanobacteria. ΔscyF also retains scytonemin production, albeit exhibiting a reduction of the production yield compared with the wild-type. This indicates that ScyF is not essential but may play an adjuvant role for scytonemin synthesis. Altogether, our findings suggest that these downstream genes are not responsible, as expected, for the late steps of scytonemin synthesis and we must look for those functions elsewhere. These findings are particularly important for biotechnological production of this sunscreen through heterologous expression of its genes in more tractable organisms. PMID:27242750

  3. Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis.

    PubMed Central

    Wiesenborn, D P; Rudolph, F B; Papoutsakis, E T

    1989-01-01

    Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site. Images PMID:2719475

  4. Characterization of five β-glycoside hydrolases from Cellulomonas fimi ATCC 484.

    PubMed

    Gao, Juan; Wakarchuk, Warren

    2014-12-01

    The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.

  5. The Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC19707

    SciTech Connect

    Klotz, M G; Arp, D J; Chain, P S; El-Sheikh, A F; Hauser, L J; Hommes, N G; Larimer, F W; Malfatti, S A; Norton, J M; Poret-Peterson, A T; Vergez, L M; Ward, B B

    2006-08-03

    The Gammaproteobacterium, Nitrosococcus oceani (ATCC 19707), is a Gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; 50.4% G+C) and a plasmid (40,420 bp) that contain 3052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. In contrast to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance and the ability to assimilate carbon via gluconeogenesis. One set of genes for type I RuBisCO was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H{sup +}-dependent F{sub 0}F{sub 1}-type, one Na{sup +}-dependent V-type).

  6. Purification and characterization of the extracellular. alpha. -amylase from Clostridium acetobutylicum ATCC 824

    SciTech Connect

    Paquet, V.; Croux, C.; Goma, G.; Soucaille, P. )

    1991-01-01

    The extracellular {alpha}-amylase (1,4-{alpha}-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (Mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying {alpha}-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the {alpha}-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. {alpha}-Amylase activity on soluble starch was optimal at pH 5.6 and 45{degree}C. The {alpha}-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a K{sub m} of 3.6 g {center dot} liter{sup {minus}1} and a K{sub cat} of 122 mol of reducing sugars {center dot} s{sup {minus}1} {center dot} mol{sup {minus}1}. The {alpha}-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the {alpha}-amylase.

  7. Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774.

    PubMed

    da Silva, Sofia Marques; Pacheco, Isabel; Pereira, Inês A Cardoso

    2012-06-01

    Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.

  8. Induction of secondary metabolism of Aspergillus terreus ATCC 20542 in the batch bioreactor cultures.

    PubMed

    Boruta, Tomasz; Bizukojc, Marcin

    2016-04-01

    Cultivation of Aspergillus terreus ATCC 20542 in a stirred tank bioreactor was performed to induce the biosynthesis of secondary metabolites and provide the bioprocess-related insights into the metabolic capabilities of the investigated strain. The activation of biosynthetic routes was attempted by the diversification of process conditions and growth media. Several strategies were tested, including the addition of rapeseed oil or inulin, changing the concentration of nitrogen source, reduction of chlorine supply, cultivation under saline conditions, and using various aeration schemes. Fifteen secondary metabolites were identified in the course of the study by using ultra-high performance liquid chromatography coupled with mass spectrometry, namely mevinolinic acid, 4a,5-dihydromevinolinic acid, 3α-hydroxy-3,5-dihydromonacolin L acid, terrein, aspulvinone E, dihydroisoflavipucine, (+)-geodin, (+)-bisdechlorogeodin, (+)-erdin, asterric acid, butyrolactone I, desmethylsulochrin, questin, sulochrin, and demethylasterric acid. The study also presents the collection of mass spectra that can serve as a resource for future experiments. The growth in a salt-rich environment turned out to be strongly inhibitory for secondary metabolism and the formation of dense and compact pellets was observed. Generally, the addition of inulin, reducing the oxygen supply, and increasing the content of nitrogen source did not enhance the production of examined molecules. The most successful strategy involved the addition of rapeseed oil to the chlorine-deficient medium. Under these conditions, the highest levels of butyrolactone I, asterric acid, and mevinolinic acid were achieved and the presence of desmethylsulochrin and (+)-bisdechlorogeodin was detected in the broth. The constant and relatively high aeration rate in the idiophase was shown to be beneficial for terrein and (+)-geodin biosynthesis. PMID:26603760

  9. gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405.

    PubMed Central

    Mäkinen, P L; Mäkinen, K K

    1997-01-01

    The human oral spirochete Treponema denticola ATCC 35405 was shown to exhibit relatively high enzyme activity toward the gamma-glutamyl amide bond present in N-gamma-L-glutamyl-4-nitroaniline. The enzyme responsible for this catalysis (gamma-glutamyltransferase [GGT]; EC 2.3.2.2) was purified by means of fast protein liquid chromatography to two sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-pure forms from a mild (0.1%) Triton X-100 extract of washed cells. The GGT was studied primarily with regard to its hydrolytic activity by using N-gamma-L-glutamyl-4-nitroaniline as a substrate, although the GGT was shown to catalyze transpeptidation reactions. The high-molecular-mass form of the GGT gave a value of about 213 kDa by SDS-PAGE when heat treatment was omitted and one of 26 kDa after heat treatment; mass spectrometry gave a value of 26.877. The larger form may represent an aggregate with nonprotein structures (possibly of a carbohydrate nature). The preliminary N-terminal sequence of the GGT is MKKPLIGITGSXLYETSQXXF. The enzyme was highly active on glutathione, transferring its Glu residue either to a water molecule or to the Gly-L-Leu dipeptide. The GGT stability was absolutely dependent on the presence of free thiol(s), while no evidence of metalloenzyme nature was obtained. The proposed location of the GGT in the outer cell envelope and its high activity on glutathione, a major nonprotein thiol present in virtually all cells, suggest that the GGT may play a role in the propagation of T. denticola within inflamed periodontal tissues. PMID:9009331

  10. Regulation of Three Nitrogenase Gene Clusters in the Cyanobacterium Anabaena variabilis ATCC 29413

    PubMed Central

    Thiel, Teresa; Pratte, Brenda S.

    2014-01-01

    The filamentous cyanobacterium Anabaena variabilis ATCC 29413 fixes nitrogen under aerobic conditions in specialized cells called heterocysts that form in response to an environmental deficiency in combined nitrogen. Nitrogen fixation is mediated by the enzyme nitrogenase, which is very sensitive to oxygen. Heterocysts are microxic cells that allow nitrogenase to function in a filament comprised primarily of vegetative cells that produce oxygen by photosynthesis. A. variabilis is unique among well-characterized cyanobacteria in that it has three nitrogenase gene clusters that encode different nitrogenases, which function under different environmental conditions. The nif1 genes encode a Mo-nitrogenase that functions only in heterocysts, even in filaments grown anaerobically. The nif2 genes encode a different Mo-nitrogenase that functions in vegetative cells, but only in filaments grown under anoxic conditions. An alternative V-nitrogenase is encoded by vnf genes that are expressed only in heterocysts in an environment that is deficient in Mo. Thus, these three nitrogenases are expressed differentially in response to environmental conditions. The entire nif1 gene cluster, comprising at least 15 genes, is primarily under the control of the promoter for the first gene, nifB1. Transcriptional control of many of the downstream nif1 genes occurs by a combination of weak promoters within the coding regions of some downstream genes and by RNA processing, which is associated with increased transcript stability. The vnf genes show a similar pattern of transcriptional and post-transcriptional control of expression suggesting that the complex pattern of regulation of the nif1 cluster is conserved in other cyanobacterial nitrogenase gene clusters. PMID:25513762

  11. Induction of secondary metabolism of Aspergillus terreus ATCC 20542 in the batch bioreactor cultures.

    PubMed

    Boruta, Tomasz; Bizukojc, Marcin

    2016-04-01

    Cultivation of Aspergillus terreus ATCC 20542 in a stirred tank bioreactor was performed to induce the biosynthesis of secondary metabolites and provide the bioprocess-related insights into the metabolic capabilities of the investigated strain. The activation of biosynthetic routes was attempted by the diversification of process conditions and growth media. Several strategies were tested, including the addition of rapeseed oil or inulin, changing the concentration of nitrogen source, reduction of chlorine supply, cultivation under saline conditions, and using various aeration schemes. Fifteen secondary metabolites were identified in the course of the study by using ultra-high performance liquid chromatography coupled with mass spectrometry, namely mevinolinic acid, 4a,5-dihydromevinolinic acid, 3α-hydroxy-3,5-dihydromonacolin L acid, terrein, aspulvinone E, dihydroisoflavipucine, (+)-geodin, (+)-bisdechlorogeodin, (+)-erdin, asterric acid, butyrolactone I, desmethylsulochrin, questin, sulochrin, and demethylasterric acid. The study also presents the collection of mass spectra that can serve as a resource for future experiments. The growth in a salt-rich environment turned out to be strongly inhibitory for secondary metabolism and the formation of dense and compact pellets was observed. Generally, the addition of inulin, reducing the oxygen supply, and increasing the content of nitrogen source did not enhance the production of examined molecules. The most successful strategy involved the addition of rapeseed oil to the chlorine-deficient medium. Under these conditions, the highest levels of butyrolactone I, asterric acid, and mevinolinic acid were achieved and the presence of desmethylsulochrin and (+)-bisdechlorogeodin was detected in the broth. The constant and relatively high aeration rate in the idiophase was shown to be beneficial for terrein and (+)-geodin biosynthesis.

  12. Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633.

    PubMed Central

    Klein, C; Entian, K D

    1994-01-01

    Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. Recently, we reported regarding genes spaB, spaT, and spaC (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Microbiol. 58:132-142, 1992) which are involved in the biosynthesis of subtilin, and genes spaR and spaK (C. Klein, C. Kaletta, and K.-D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), which regulate subtilin biosynthesis via a histidine kinase/response regulator system. Further sequence analysis revealed the presence of three additional open reading frames, spaI, spaF, and spaG, downstream of the structural gene spaS. The spaI gene encodes a hydrophilic 19.3-kDa lipoprotein containing a consensus signal sequence, indicating that this protein might be membrane anchored. A similar gene, nisI, has been identified in the nisin producer. SpaF shows strong homology to members of the family of ABC transporters. spaG encodes a hydrophobic protein which might form the active transporter together with SpaF. Gene disruption mutants in all three genes were still able to produce subtilin; however, these mutants were more sensitive to subtilin than the wild-type strain. These results show that these genes are involved in the immunity mechanism of the producer strain. A similar involvement of an ABC transporter in the self-protection mechanism has been described for the McbE and McbF transporter, which confers immunity against microcin B17 in Escherichia coli. Mutants containing mutations in the genes spaR and spaK, which are responsible for regulation of subtilin biosynthesis, also became more sensitive to subtilin.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8085823

  13. Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC 19707†

    PubMed Central

    Klotz, Martin G.; Arp, Daniel J.; Chain, Patrick S. G.; El-Sheikh, Amal F.; Hauser, Loren J.; Hommes, Norman G.; Larimer, Frank W.; Malfatti, Stephanie A.; Norton, Jeanette M.; Poret-Peterson, Amisha T.; Vergez, Lisa M.; Ward, Bess B.

    2006-01-01

    The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H+-dependent F0F1 type, one Na+-dependent V type). PMID:16957257

  14. Immobilization of Lactobacillus salivarius ATCC 11741 on loofa sponge coated with chitosan for lactic acid fermentation.

    PubMed

    Chantawongvuti, Ratchat; Veerajetbodithat, Juntanee; Jaturapiree, Phimchanok; Muangnapoh, Chirakarn

    2010-01-01

    Lactic acid (LA) fermentation by Lactobacillus salivarius ATCC 11741 immobilized on loofa sponge (LS) was evaluated. To increase the surface area of LS for cell immobilization, H2O2 and chitosan were introduced as surface modifying reagents. Four chitosan of different molecular weight were separately coated on LS. All experiments were conducted in shaking flask mode at 100 rpm rotating speed and 37 degrees with 5% CaCO3 as a pH regulating agent. The effects of initial glucose concentration were investigated in the range of 20-100 g L-1 on LA fermentation by free cells. The results indicate that the maximum concentration of LA was produced with 50 g L-1 glucose concentration. The immobilized cell system produced 1.5 times higher concentration than free cells for 24 h of fermentation. Moreover, immobilized cells can shorten the fermentation time by 2 fold compared with free cells at the same level of LA concentration. At 1% w/v chitosan in 2% v/v acetic acid, Yp/s and productivities of various molecular weight of chitosan were insignificantly different. Repeated batch fermentations showed 5 effective recycles with Yp/s and productivity in the range of 0.55-0.85 and 0.90-1.20 g L-1 h-1, respectively. It is evident that immobilization of L. salivarius onto LS permits reuse of the system under these fermentation conditions. Scanning electron micrographs indicated that there were more intact cells on the chitosan-treated LS than on the untreated LS, thus confirming the effectiveness of the LS-chitosan combination when being utilized as a promising immobilization carrier for LA fermentation. PMID:20134241

  15. Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

    PubMed Central

    Gao, Juan

    2014-01-01

    The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose. PMID:25225266

  16. Streptococcus salivarius ATCC 25975 possesses at least two genes coding for primer-independent glucosyltransferases.

    PubMed Central

    Simpson, C L; Giffard, P M; Jacques, N A

    1995-01-01

    Fractionation of the culture medium showed that Streptococcus salivarius ATCC 25975 secreted a glucosyltransferase (Gtf) that was primer independent. On the basis of this observation, a gene library of S. salivarius chromosomal DNA cloned into lambda L47.1 was screened for a gene(s) coding for such an activity. As a result of this screening process, two new gtf genes, gtfL and gtfM, both of which coded for primer-independent Gtf activities, were isolated. GtfL produced an insoluble glucan that was refractory to digestion by the endo-(1-->6)-alpha-D-glucanase. of Chaetonium gracile, while GtfM produced a soluble glucan that was readily degraded by the glucanase. Comparison of the deduced amino acid sequences of gtfL and gtfM with 10 other available Gtf sequences allowed the relatedness of the conserved catalytic regions to be assessed. This analysis showed that the 12 enzymes did not form clusters based on their primer dependencies or on their product solubilities. Further analysis of the YG repeats in the C-terminal glucan-binding domains of GtfJ, GtfK, GtfL, and GtfM from S. salivarius showed that there was strong homology between a block of contiguous triplet YG repeats present in the four alleles. These blocks of YG repeats were coded for by a region of each gene that appeared to have arisen as a result of a recent duplication event(s). PMID:7822030

  17. Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC 19707

    SciTech Connect

    Klots, Martin G.; Arp, D J; Chain, Patrick S; El-Sheikh, Amal F.; Hauser, Loren John; Hommes, Norman G.; Larimer, Frank W; Malfatti, Stephanie; Norton, Jeanette M.; Poret-Peterson, Amisha T.; Vergez, Lisa; Ward, Bess B.

    2006-01-01

    The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H+-dependent F0F1 type, one Na+-dependent V type).

  18. Multicopy Integration and Expression of Heterologous Genes in Methylobacterium extorquens ATCC 55366†

    PubMed Central

    Choi, Young J.; Bourque, Denis; Morel, Lyne; Groleau, Denis; Míguez, Carlos B.

    2006-01-01

    High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (PmxaF) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [β-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens. PMID:16391115

  19. Activation of dormant bacterial genes by Nonomuraea sp. strain ATCC 39727 mutant-type RNA polymerase.

    PubMed

    Talà, Adelfia; Wang, Guojun; Zemanova, Martina; Okamoto, Susumu; Ochi, Kozo; Alifano, Pietro

    2009-02-01

    There is accumulating evidence that the ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to the presence of cryptic gene clusters. The activation of dormant genes is therefore one of the most important areas of experimental research for the discovery of drugs in these organisms. The recent observation that several actinomycetes possess two RNA polymerase beta-chain genes (rpoB) has opened up the possibility, explored in this study, of developing a new strategy to activate dormant gene expression in bacteria. Two rpoB paralogs, rpoB(S) and rpoB(R), provide Nonomuraea sp. strain ATCC 39727 with two functionally distinct and developmentally regulated RNA polymerases. The product of rpoB(R), the expression of which increases after transition to stationary phase, is characterized by five amino acid substitutions located within or close to the so-called rifampin resistance clusters that play a key role in fundamental activities of RNA polymerase. Here, we report that rpoB(R) markedly activated antibiotic biosynthesis in the wild-type Streptomyces lividans strain 1326 and also in strain KO-421, a relaxed (rel) mutant unable to produce ppGpp. Site-directed mutagenesis demonstrated that the rpoB(R)-specific missense H426N mutation was essential for the activation of secondary metabolism. Our observations also indicated that mutant-type or duplicated, rpoB often exists in nature among rare actinomycetes and will thus provide a basis for further basic and applied research.

  20. Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions.

    PubMed

    Alagesan, Swathi; Gaudana, Sandeep B; Sinha, Avinash; Wangikar, Pramod P

    2013-11-01

    Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

  1. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    PubMed Central

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-01-01

    Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR. PMID:26249707

  2. Mutational Studies of Putative Biosynthetic Genes for the Cyanobacterial Sunscreen Scytonemin in Nostoc punctiforme ATCC 29133.

    PubMed

    Ferreira, Daniela; Garcia-Pichel, Ferran

    2016-01-01

    The heterocyclic indole-alkaloid scytonemin is a sunscreen found exclusively among cyanobacteria. An 18-gene cluster is responsible for scytonemin production in Nostoc punctiforme ATCC 29133. The upstream genes scyABCDEF in the cluster are proposed to be responsible for scytonemin biosynthesis from aromatic amino acid substrates. In vitro studies of ScyA, ScyB, and ScyC proved that these enzymes indeed catalyze initial pathway reactions. Here we characterize the role of ScyD, ScyE, and ScyF, which were logically predicted to be responsible for late biosynthetic steps, in the biological context of N. punctiforme. In-frame deletion mutants of each were constructed (ΔscyD, ΔscyE, and ΔscyF) and their phenotypes studied. Expectedly, ΔscyE presents a scytoneminless phenotype, but no accumulation of the predicted intermediaries. Surprisingly, ΔscyD retains scytonemin production, implying that it is not required for biosynthesis. Indeed, scyD presents an interesting evolutionary paradox: it likely originated in a duplication event from scyE, and unlike other genes in the operon, it has not been subjected to purifying selection. This would suggest that it is a pseudogene, and yet scyD is highly conserved in the scytonemin operon of cyanobacteria. ΔscyF also retains scytonemin production, albeit exhibiting a reduction of the production yield compared with the wild-type. This indicates that ScyF is not essential but may play an adjuvant role for scytonemin synthesis. Altogether, our findings suggest that these downstream genes are not responsible, as expected, for the late steps of scytonemin synthesis and we must look for those functions elsewhere. These findings are particularly important for biotechnological production of this sunscreen through heterologous expression of its genes in more tractable organisms. PMID:27242750

  3. A Model of Cyclic Transcriptomic Behavior in Cyanobacterium Cyanothece sp. ATCC 51142

    SciTech Connect

    McDermott, Jason E.; Oehmen, Christopher S.; McCue, Lee Ann; Hill, Eric A.; Choi, Daniel M.; Stockel, Jana; Liberton, Michelle L.; Pakrasi, Himadri B.; Sherman, Louis A.

    2011-07-01

    Systems biology attempts to reconcile large amounts of disparate data with existing knowledge to provide models of functioning biological systems. Useful and predictive models aim to summarize complex and dynamic processes and represent the relationships between these processes. The cyanobacterial Cyanothece species Strain sp. ATCC 51142 is an excellent candidate for such systems studies because: (i) it displays tight functional regulation as it must separate the opposing processes of oxygen-generating photosynthesis and oxygen-sensitive nitrogen fixation temporally in the same cell, ; (ii) it has robust cyclic patterns at the genetic, protein and metabolomic levels, ; and (iii) and it has potential applications for bioenergy and carbon sequestration, and thus a predictive model of its function is of practical use. We have represented the transcriptomic data from Cyanothece 51142 under diurnal light/dark cycles as a high-level functional abstraction and describe development of a predictive in silico model of diurnal and circadian behavior in terms of regulatory and metabolic processes in Cyanothece 51142. Our model provides a way to integrate disparate data types into a framework that can be used to explain behavior, generate high-quality predictions for validation, and to suggest future experiments. We show that incorporating network topology into the model improves performance in terms of our ability to explain the behavior of the system under new conditions. The model presented robustly describes transcriptomic behavior of Cyanothece 51142 under different cyclic and non-cyclic growth conditions robustly, and represents a significant advance in the understanding of gene regulation in this important organism.

  4. Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

    SciTech Connect

    Andersen, Mikael R.; Salazar, Margarita; Schaap, Peter; van de Vondervoort, Peter; Culley, David E.; Thykaer, Jette; Frisvad, Jens C.; Nielsen, Kristian F.; Albang, Richard; Albermann, Kaj; Berka, Randy; Braus, Gerhard; Braus-Stromeyer, Susanna A.; Corrochano, Luis; Dai, Ziyu; van Dijck, Piet; Hofmann, Gerald; Lasure, Linda L.; Magnuson, Jon K.; Menke, Hildegard; Meijer, Martin; Meijer, Susan; Nielsen, Jakob B.; Nielsen, Michael L.; van Ooyen, Albert; Pel, Herman J.; Poulsen, Lars; Samson, Rob; Stam, Hein; Tsang, Adrian; van den Brink, Johannes M.; ATkins, Alex; Aerts, Andrea; Shapiro, Harris; Pangilinan, Jasmyn; Salamov, Asaf; Lou, Yigong; Lindquist, Erika; Lucas, Susan; Grimwood, Jane; Grigoriev, Igor V.; Kubicek, Christian P.; Martinez, Diego; van Peij, Noel; Roubos, Johannes A.; Nielsen, Jens B.; Baker, Scott E.

    2011-06-01

    The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases and protein transporters.

  5. Proline iminopeptidase from the outer cell envelope of the human oral spirochete Treponema denticola ATCC 35405.

    PubMed Central

    Mäkinen, K K; Chen, C Y; Mäkinen, P L

    1996-01-01

    Certain periodontopathic organisms have been shown to exhibit high activity of proline iminopeptidase (PIPase). The human oral spirochete Treponema denticola ATCC 35405 was found to contain an easily extractable, novel PIPase (EC 3.4.11.5), which was purified to a sodium dodecyl sulfate- polyacrylamide gel electrophoresis-pure form by means of fast protein liquid chromatographic procedures. The range of the minimum monomeric molecular mass (280 amino acid residues) of the PIPase, based on amino acid analysis, was 30.35 to 30.39 kDa, but the likely in vivo form of the enzyme is a tetramer (minimum mass, 120.2 to 120.4 kDa). The molecular masses based on laser desorption mass spectrometry were 36.058 kDa for the monomer and 72.596 kDa for a dimer. The PIPase cleaves specifically the Pro-Y bond in dipeptides where Y is preferably Arg or Lys. Pro-Gln, Pro-Asn, and Pro-Ala were also good substrates, while Pro-Glu was hydrolyzed slowly and Pro-Asp was not hydrolyzed at all. Tripeptides were poor substrates or were not hydrolyzed (an exception was Pro-Gly-Gly, which cleaved at a moderate rate). Larger molecules, such as poly-L-Pro, were not hydrolyzed. The T. denticola enzyme can be regarded as a true PIPase, since replacing Pro in Pro-Y with other amino acid residues resulted in no hydrolysis. The activity of the PIPase may depend on an active carboxyl group and on an active seryl residue but not on metal cations. Diethylpyrocarbonate inactivated the enzyme in a reaction that was not reversible upon addition of NH2OH. The enzyme contains a relatively large percentage (ca. 15%) of proline residues. The dominance of the PIPase activity among aminopeptidase activities present in T. denticola and the proposed location of the enzyme in the outer cell envelope suggest that it has a vital function in the propagation of the cells within their biological niche (inflamed human periodontal tissues). The biologic role of the PIPase may be envisaged as in the termination of the

  6. Transcriptomic and genomic analysis of cellulose fermentation by Clostridium thermocellum ATCC 27405

    SciTech Connect

    Raman, Babu; McKeown, Catherine K; Rodriguez, Jr., Miguel; Brown, Steven D; Mielenz, Jonathan R

    2011-01-01

    The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. A time-series analysis of gene expression revealed changes in transcript levels of {approx}40% of genes ({approx}1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide hydrolysis products

  7. Characterizations of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

    SciTech Connect

    Chai,S.; Ju, T.; Dang, M.; Goldsmith, R.; Maroney, M.; Pochapsky, T.

    2008-01-01

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1, 2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M2+ metal ion bound in the active site. The Ni2+-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe2+-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates metal in vivo but

  8. Characterization of Metal Binding in the Active Sites of acireductone dioxygenase Isoforms from Klebsiella ATCC 8724

    SciTech Connect

    S Chai; T Ju; M Dang; R Goldsmith; M Maroney; T Pochapsky

    2011-12-31

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M{sup 2+} metal ion bound in the active site. The Ni{sup 2+}-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe{sup 2+}-bound FeARD catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates

  9. Interaction of Wild Strains of Aspergilla with Aspergillus parasiticus ATCC15517 and Aflatoxin Production †

    PubMed Central

    Martins, H. Marina; Almeida, Inês; Marques, Marta; Bernardo, Fernando

    2008-01-01

    Aflatoxins are secondary metabolites produced by some competent mould strains of Aspergillus flavus, A. parasiticus and A. nomius. These compounds have been extensively studied with regards to their toxicity for animals and humans; they are able to induce liver cancer and may cause a wide range of adverse effects in living organisms. Aflatoxins are found as natural contaminants of food and feed; the main line of the strategy to control them is based on the prevention of the mould growth in raw vegetable or during its storage and monitoring of each crop batch. Mould growth is conditioned by many ecological factors, including biotic ones. Hazard characterization models for aflatoxins in crops must take into consideration biotic interactions between moulds and their potential effects on growth development. The aim of this work is to study the effect of the biotic interaction of 14 different wild strains of Aspergilla (different species), with a competent strain (Aspergillus parasiticus ATCC 15517) using an in vitro production model. The laboratory model used was a natural matrix (humidified cracked corn), on which each wild strain challenged the aflatoxin production of a producer strain. Cultures were incubated at 28°C for 12 days and sampled at the 8th and 12th. Aflatoxin detection and quantification was performed by HPLC using a procedure with a MRPL = 1 μg/kg. Results of those interactive cultures revealed both synergic and antagonistic effects on aflatoxin biosynthesis. Productivity increases were particularly evident on the 8th day of incubation with wild strains of A. flavipes (+ 70.4 %), A. versicolor (+ 54.9 %) and A. flavus 3 (+ 62.6 %). Antagonistic effects were found with A. niger (− 69.5%), A. fumigatus (− 47.6 %) and A. terreus (− 47.6 %) on the 12th day. The increased effects were more evident on the 8th of incubation and the decreases were more patent on the 12th day. Results show that the development of Aspergilla strains concomitantly with

  10. Design and production of functionalized biopolyesters by Methylobacterium extorquens ATCC 55366: Toward new tissue engineering materials

    NASA Astrophysics Data System (ADS)

    Hoefer, Heinrich Friedrich Philipp Till Nikolaus

    Vascular networks are required to support the formation and function of three-dimensional tissues. Biodegradable scaffolds are being considered in order to promote vascularization where natural regeneration of lost or destroyed vascular networks fails. Particularly; composite materials are expected to fulfill the complex demands of a patient's body to support wound healing. Microbial biopolyesters are being regarded as such second and third generation biomaterials. Methylobacterium extorquens is one of several microorganisms that should be considered for the production of advanced polyhydroxyalkanoates (PHAs). M. extorquens displays a distinct advantage in that it is able to utilize methanol as an inexpensive substrate for growth and biopolyester production. The design of functionalized PHAs, which would be made of both saturated short-chain-length (scl, C ≤ 5) and unsaturated medium-chain-length (mcl, 6 ≤ C ≤ 14) monomeric units, aimed at combining desirable material properties of inert scl/mcl-PHAs with those of functionalized mcl-PHAs. By independently inserting the phaC1 or the phaC2 gene from Pseudomonas fluorescens GK13, recombinant M. extorquens strains were obtained which were capable of producing PHAs containing C-C double bonds. A fermentation process was developed to obtain gram quantities of biopolyesters employing the recombinant M. extorquens ATCC 55366 strain which harbored the phaC2 gene of P. fluorescens GK13, the better one of the two strains at incorporating unsaturated monomeric units. The PHAs produced were found in a blend of scl-PHAs and functionalized scl/mcl-PHAs (4 ≤ C ≤ 6), which were the products of the native and of the recombinant PHA synthase, respectively. Thermo-mechanical analysis confirmed that the functionalized scl/mcl-PHAs exhibited the desirable material properties expected. This project contributed to current research on polyhydroxyalkanoates at different levels. The terminal double bonds of the functionalized scl

  11. Inhibition of Listeria monocytogenes ATCC 19115 on ham steak by tea bioactive compounds incorporated into chitosan-coated plastic films

    PubMed Central

    2012-01-01

    Background The consumer demands for better quality and safety of food products have given rise to the development and implementation of edible films. The use of antimicrobial films can be a promising tool for controlling L. monocytogenes on ready to eat products. The aim of this study was to develop effective antimicrobial films incorporating bioactive compounds from green and black teas into chitosan, for controlling L. monocytogenes ATCC 19115 on vacuum-packaged ham steak. The effectiveness of these antimicrobial films was evaluated at room temperature (20°C) for 10 days and at refrigerated temperature (4°C) for 8 weeks. Results The HPLC results clearly show that relative concentrations of catechins and caffeine in green tea ranked EGCG>EGC>CAF>ECG>EC>C while in black tea extracts ranked CAF>EGCG>ECG>EGC>EC>C. The chitosan-coated plastic films incorporating green tea and black tea extracts shows specific markers identified by FTIR. Incorporating natural extracts into chitosan showed that the growth of L monocytogenes ATCC 19115 was inhibited. The efficacy of antimicrobial effect of tea extracts incorporated into chitosan-coated plastic film was dose dependent. However, chitosan-coated films without addition of tea extracts did not inhibit the growth of L. monocytogenes ATCC 19115. Chitosan-coated plastic films incorporating 4% Green tea extract was the most effective antimicrobial, reducing the initial counts from 3.2 to 2.65 log CFU/cm2 during room temperature storage and from 3.2 to 1–1.5 log CFU/cm2 during refrigerated storage. Conclusions Incorporation of tea extracts into the chitosan-coated films considerably enhanced their effectiveness against L. monocytogenes ATCC 19115. 4% Green tea incorporated into chitosan-coated plastic film had a better antilisterial effect than 2% green tea or 2% and 4% black tea. Data from this study would provide new formulation options for developing antimicrobial packaging films using tea extracts to improve the

  12. Isolation, Sequence Analysis, and Comparison of Two Plasmids (28 and 29 Kilobases) from the Biomining Bacterium Leptospirillum ferrooxidans ATCC 49879

    PubMed Central

    Coram, Nicolette J.; van Zyl, Leonardo J.; Rawlings, Douglas E.

    2005-01-01

    Two plasmids, of 28,878 bp and 28,012 bp, were isolated from Leptospirillum ferrooxidans ATCC 49879. Altogether, a total of 67 open reading frames (ORFs) were identified on both plasmids, of which 32 had predicted products with high homology to proteins of known function, while 11 ORFs had predicted products with homology to previously identified proteins of unknown function. Twenty-four ORFs had products with no homologues in the GenBank/NCBI database. An analysis of the ORFs and other features of the two plasmids, the first to be isolated from a bacterium of the genus Leptospirillum, is presented. PMID:16269793

  13. Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

    SciTech Connect

    Grigoriev, Igor V.; Baker, Scott E.; Andersen, Mikael R.; Salazar, Margarita P.; Schaap, Peter J.; Vondervoot, Peter J.I. van de; Culley, David; Thykaer, Jette; Frisvad, Jens C.; Nielsen, Kristen F.; Albang, Richard; Albermann, Kaj; Berka, Randy M.; Braus, Gerhard H.; Braus-Stromeyer, Susanna A.; Corrochano, Luis M.; Dai, Ziyu; Dijck, Piet W.M. van; Hofmann, Gerald; Lasure, Linda L.; Magnusson, Jon K.; Meijer, Susan L.; Nielsen, Jakob B.; Nielsen, Michael L.; Ooyen, Albert J.J. van; Panther, Kathyrn S.; Pel, Herman J.; Poulsen, Lars; Samson, Rob A.; Stam, Hen; Tsang, Adrian; Brink, Johannes M. van den; Atkins, Alex; Aerts, Andrea; Shapiro, Harris; Pangilinan, Jasmyn; Salamov, Asaf; Lou, Yigong; Lindquist, Erika; Lucas, Susan; Grimwood, Jane; Kubicek, Christian P.; Martinez, Diego; Peij, Noel N.M.E. van; Roubos, Johannes A.; Nielsen, Jens

    2011-04-28

    The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up-regulation of genes relevant to glucoamylase A production, such as tRNA-synthases and protein transporters. Our results and datasets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.[Supplemental materials (10 figures, three text documents and 16 tables) have been made available

  14. Characterization of Methylglyoxal Synthase from Clostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

    PubMed Central

    Huang, Ke-xue; Rudolph, Frederick B.; Bennett, George N.

    1999-01-01

    A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km and Vmax values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min−1 μg−1, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced. PMID:10388730

  15. Oral immunization of pigs with viable or inactivated Actinobacillus pleuropneumoniae serotype 9 induces pulmonary and systemic antibodies and protects against homologous aerosol challenge.

    PubMed

    Hensel, A; Stockhofe-Zurwieden, N; Petzoldt, K; Lubitz, W

    1995-08-01

    A dose-defined aerosol infection of pigs was used to study the immunogenic and protective potentials of oral immunization with dead or live Actinobacillus pleuropneumoniae serotype 9 reference strain CVI 13261 against an aerogenic challenge. Pigs were vaccinated with a single dose of 10(11) CFU of viable (n = 8) or inactivated (n = 8) A. pleuropneumoniae given orally in a gelatin capsule. After 3 weeks, vaccinated pigs and nonvaccinated controls were challenged aerogenically with a dose of 10(8) CFU of A. pleuropneumoniae CVI 13261. The protective efficacy of oral immunization was evaluated by clinical and postmortem examinations. Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples for assessment of local antibodies. Isotype-specific antibody responses in sera and in bronchoalveolar lavage fluids were determined by enzyme-linked immunosorbent assays based on whole-cell antigen. Oral immunization did not induce clinical side effects. After aerosol challenge, two animals of both vaccinated groups (25% in each case) showed a moderate fever for 2 days, whereas all four pigs (100%) of the nonvaccinated control group developed severe fever. In contrast to the controls, which developed severe pleuropneumonia, the vaccinated pigs had only mild pulmonary lesions. Three weeks after challenge, 13 of 16 vaccinated pigs (81%) were found to be free of pathomorphological changes of the lungs. From two of these pigs immunized with live bacteria we were able to reisolate A. pleuropneumoniae. A significant systemic and pulmonary increase in the concentrations of immunoglobulin A (IgA), IgM, and IgG antibodies reactive with A. pleuropneumoniae was detectable after aerosol challenge in both vaccinated groups. Immunization with viable bacteria was found to induce significantly higher concentrations of each Ig isotype in bronchoalveolar lavage fluids and sera than immunization with inactivated A. pleuropneumoniae. These serological findings were

  16. Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures.

    PubMed Central

    Nishihara, T; Ueda, N; Amano, K; Ishihara, Y; Hayakawa, H; Kuroyanagi, T; Ohsaki, Y; Nagata, K; Noguchi, T

    1995-01-01

    The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system. When mouse bone marrow cells were cultured with A. actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed. The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine. In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A. actinomycetemcomitans Y4 capsular-like polysaccharide. However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation. IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A. actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures. The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y4 capsular-like polysaccharide. These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. We sought to determine whether osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004. The formation of osteoclast-like cells was suppressed by H8 and HA1004. These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A

  17. The ferripyoverdine receptor FpvA of Pseudomonas aeruginosa PAO1 recognizes the ferripyoverdines of P. aeruginosa PAO1 and P. fluorescens ATCC 13525.

    PubMed

    Meyer, J M; Stintzi, A; Poole, K

    1999-01-01

    FpvA, the ferripyoverdine outer membrane receptor of Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is not specific to the pyoverdine produced by PAO1, but is also able to recognize the structurally different (ferri)pyoverdine of P. fluorescens ATCC 13525. The specificity of FpvA was assessed by iron uptake competitions using the wild-type strains P. aeruginosa ATCC 15692 and P. fluorescens ATCC 13525 and their respective ferripyoverdines, and by fpvA gene complementation of a FpvA-deficient mutant of P. aeruginosa ATCC 15692. The receptor mutant was able to utilize none of the two pyoverdines, while the same but fpvA-complemented mutant recovered simultaneously the ability to incorporate iron thanks to each of the two siderophores. The broad specificity of recognition of FpvA is viewed as an advantage for the strain in iron competition. Moreover, it allows an interesting approach for the understanding of the recognition mechanism between a (ferri)pyoverdine and its cognate outer membrane receptor. PMID:9919663

  18. Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: A receptor for lectin-mediated interbacterial adherence

    SciTech Connect

    Abeygunawardana, C.; Bush, C.A. ); Cisar, J.O. )

    1991-07-02

    Lectin-carbohydrate binding is known to play an important role in a number of different cell-cell interactions including those between certain species of oral streptococci and actinomyces that colonize teeth. The cell wall polysaccharides of Streptococcus oralis ATCC 10557, S. oralis 34, and Streptococcus mitis J22, although not identical antigenically, each function as a receptor molecule for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii. Carbohydrate analysis of the receptor polysaccharide isolated from S. oralis ATCC 10557 shows galactose (3 mol), glucose (1 mol), GalNAc (1 mol), and rhamnose (1 mol). {sup 1}H NMR spectra of the polysaccharide show that is partially O-acetylated. Analysis of the {sup 1}H NMR spectrum of the de-O-acetylated polysaccharide shows that it is composed of repeating subunits containing six monosaccharides and that the subunits are joined by a phosphodiester linkage. The {sup 1}H and {sup 13}C NMR spectra were completely assigned by two-dimensional homonuclear correlation methods and by {sup 1}H-detected heteronuclear multiple-quantum correlation ({sup 1}H({sup 13}C)HMQC). The complete {sup 1}H and {sup 13}C assignment of the native polysaccharide was carried out by the same techniques augmented by a {sup 13}C-coupled hybrid HMQC-COSY method, which is shown to be especially useful for carbohydrates in which strong coupling and overlapping peaks in the {sup 1}H spectrum pose difficulties.

  19. Inactivation of Listeria monocytogenes ATCC 7644 on fresh-cut tomato using nisin in combinations with organic salts.

    PubMed

    Oladunjoye, Adebola O; Singh, Suren; Ijabadeniyi, Oluwatosin A

    2016-01-01

    The inhibition of Listeria monocytogenes ATCC 7644 on fresh-cut tomato was investigated using nisin alone, and in combinations with organic salts. Nisin at a concentration of 5000UI/mL was introduced alone or in combination with an organic salt (sodium citrate or sodium acetate each at 3 and 5g/100mL each) on fresh-cut tomato previously inoculated with 10(8)CFU/mL of L. monocytogenes ATCC 7644. Chlorine at 200ppm was used as a control. The inoculated samples were incubated at different temperatures (4, 10 and 25°C) and examined at 0, 24, 48 and 72h. The effects of the antimicrobial treatments on quality parameters of tomato (pH, soluble solids, titratable acidity and vitamin C) were also evaluated, and colour parameters were observed at the lowest storage temperature for 10 days. Both nisin and the organic salts inhibited growth of L. monocytogenes, but the combinations of two compounds were more effective. The nisin-sodium citrate (5%) combination was significantly (p≤0.05) effective, while chlorine was least effective against L. monocytogenes. The quality parameters were substantially retained, especially at 4°C, suggesting good shelf stability at a low temperature. These results substantiate the use of the cheap and eco-friendly approach to reducing this pathogen of health concern in common fresh produce. PMID:27261167

  20. Antiserum against Raoultella terrigena ATCC 33257 identifies a large number of Raoultella and Klebsiella clinical isolates as serotype O12.

    PubMed

    Mertens, Katja; Müller-Loennies, Sven; Stengel, Petra; Podschun, Rainer; Hansen, Dennis S; Mamat, Uwe

    2010-12-01

    Raoultella terrigena ATCC 33257, recently reclassified from the genus Klebsiella, is a drinking water isolate and belongs to a large group of non-typeable Klebsiella and Raoultella strains. Using an O-antiserum against a capsule-deficient mutant of this strain, we could show a high prevalence (10.5%) of the R. terrigena O-serotype among non-typeable, clinical Klebsiella and Raoultella isolates. We observed a strong serological cross-reaction with the K. pneumoniae O12 reference strain, indicating that a large percentage of these non-typeable strains may belong to the O12 serotype, although these are currently not detectable by the K. pneumoniae O12 reference antiserum in use. Therefore, we analyzed the O-polysaccharide (O-PS) structure and genetic organization of the wb gene cluster of R. terrigena ATCC 33257, and both confirmed a close relation of R. terrigena and K. pneumoniae O12. The two strains possess an identical O-PS, lipopolysaccharide core structure, and genetic organization of the wb gene cluster. Heterologous expression of the R. terrigena wb gene cluster in Escherichia coli K-12 resulted in the WecA-dependent synthesis of an O-PS reactive with the K. pneumoniae O12 antiserum. The serological data presented here suggest a higher prevalence of the O12-serotype among Klebsiella and Raoultella isolates than generally assumed.

  1. Efficient Simultaneous Saccharification and Fermentation of Inulin to 2,3-Butanediol by Thermophilic Bacillus licheniformis ATCC 14580

    PubMed Central

    Li, Lixiang; Chen, Chao; Li, Kun; Wang, Yu; Gao, Chao; Ma, Cuiqing

    2014-01-01

    2,3-Butanediol (2,3-BD) is an important starting material for the manufacture of bulk chemicals. For efficient and large-scale production of 2,3-BD through fermentation, low-cost substrates are required. One such substrate, inulin, is a polydisperse fructan found in a wide variety of plants. In this study, a levanase with high inulinase activity and high pH and temperature stability was identified in Bacillus licheniformis strain ATCC 14580. B. licheniformis strain ATCC 14580 was found to efficiently produce 2,3-BD from fructose at 50°C. Then, the levanase was used for simultaneous saccharification and fermentation (SSF) of inulin to 2,3-BD. A fed-batch SSF yielded 103.0 g/liter 2,3-BD in 30 h, with a high productivity of 3.4 g/liter · h. The results suggest that the SSF process developed with the thermophilic B. licheniformis strain used might be a promising alternative for efficient 2,3-BD production from the favorable substrate inulin. PMID:25107977

  2. Growth and nitrite and nitrous oxide accumulation of Paracoccus denitrificans ATCC 19367 in the presence of selected pesticides.

    PubMed

    Sáez, Florentina; Pozo, Clementina; Gómez, Miguel Angel; Rodelas, Belén; Gónzalez-López, Jesús

    2003-09-01

    The effects of the application of eight pesticides (aldrin, lindane, dimetoate, methylparathion, methidation, atrazine, simazine, and captan) on growth, respiratory activity (as CO2 production), denitrifying activity (as N2O released), and nitrite accumulation in the culture medium by Paracoccus denitrificans strain ATCC 19367 were studied. The fungicide captan totally inhibited growth and biological activity of P. denitrificans, while the rest of the tested pesticides delayed the growth and CO2 release of P. denitrificans but did not drastically affect the bacterial growth or respiratory capacity after 96 h of culture. The denitrifying activity of P. denitrificans ATCC 19367 (as N2O released) was negatively affected by all tested pesticides. The release of N2O was strongly inhibited by several organochlorinated and organophosphorated insecticides (aldrin, lindane, dimetoate, and methidation), which led to high accumulation of nitrite in the surrounding medium. Atrazine decreased N2O release after 48 h of culture because of negative effects on growth, and methylparathion and simazine delayed the onset of N2O release by P. denitrificans. These three pesticides reduced the accumulation of NO2- compared to unamended control cultures. PMID:12959522

  3. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

    PubMed

    Fujiwara, T; Fukumori, Y

    1996-04-01

    A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. PMID:8606159

  4. Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142.

    PubMed Central

    Schneegurt, M A; Sherman, D M; Nayar, S; Sherman, L A

    1994-01-01

    It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms. Images PMID:8132452

  5. Formation of biofilm by Listeria monocytogenes ATCC 19112 at different incubation temperatures and concentrations of sodium chloride.

    PubMed

    Lee, H Y; Chai, L C; Pui, C F; Mustafa, S; Cheah, Y K; Nishibuchi, M; Radu, S

    2013-01-01

    Biofilm formation can lead to various consequences in the food processing line such as contamination and equipment breakdowns. Since formation of biofilm can occur in various conditions; this study was carried out using L. monocytogenes ATCC 19112 and its biofilm formation ability tested under various concentrations of sodium chloride and temperatures. Cultures of L. monocytogenes ATCC 19112 were placed in 96-well microtitre plate containing concentration of sodium chloride from 1-10% (w/v) and incubated at different temperature of 4 °C, 30 °C and 45 °C for up to 60 h. Absorbance reading of crystal violet staining showed the density of biofilm formed in the 96-well microtitre plates was significantly higher when incubated in 4 °C. The formation of biofilm also occurs at a faster rate at 4 °C and higher optical density (OD 570 nm) was observed at 45 °C. This shows that storage under formation of biofilm that may lead to a higher contamination along the processing line in the food industry. Formation of biofilm was found to be more dependent on temperature compared to sodium chloride stress.

  6. Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation.

    PubMed

    Chatterjee, Joyee; Giri, Sudipta; Maity, Sujan; Sinha, Ankan; Ranjan, Ashish; Rajshekhar; Gupta, Suvroma

    2015-01-01

    Proteases are the most important group of enzymes utilized commercially in various arenas of industries, such as food, detergent, leather, dairy, pharmaceutical, diagnostics, and waste management, accounting for nearly 20% of the world enzyme market. Microorganisms of specially Bacillus genera serve as a vast repository of diverse set of industrially important enzymes and utilized for the large-scale enzyme production using a fermentation technology. Approximately 30%-40% of the cost of industrial enzymes originates from the cost of the growth medium. This study is attempted to produce protease from Bacillus subtilis (ATCC 6633) after optimization of various process parameters with the aid of solid-state fermentation using a cheap nutrient source such as wheat bran. B. subtilis (ATCC 6633) produces proteases of molecular weight 36 and 20 kDa, respectively, in the fermented medium as evident from SDS zymogram. Alkaline protease activity has been detected with optimum temperature at 50 °C and is insensitive to ethylenediaminetetraacetic acid. This thermostable alkaline protease exhibits dual pH optimum at 7 and 10 with moderate pH stability at alkaline pH range. It preserves its activity in the presence of detergent such as SDS, Tween 20, and Triton X-100 and may be considered as an effective additive to detergent formulation with some industrial importance.

  7. A Novel Metallo-β-Lactamase Involved in the Ampicillin Resistance of Streptococcus pneumoniae ATCC 49136 Strain

    PubMed Central

    Chang, Chia-Yu; Lin, Hui-Jen; Li, Yaw-Kuen

    2016-01-01

    Streptococcus pneumoniae, a penicillin-sensitive bacterium, is recognized as a major cause of pneumonia and is treated clinically with penicillin-based antibiotics. The rapid increase in resistance to penicillin and other antibiotics affects 450 million people globally and results in 4 million deaths every year. To unveil the mechanism of resistance of S. pneumoniae is thus an important issue to treat streptococcal disease that might consequently save millions of lives around the world. In this work, we isolated a streptococci-conserved L-ascorbate 6-phosphate lactonase, from S. pneumoniae ATCC 49136. This protein reveals a metallo-β-lactamase activity in vitro, which is able to deactivate an ampicillin-based antibiotic by hydrolyzing the amide bond of the β-lactam ring. The Michaelis parameter (Km) = 25 μM and turnover number (kcat) = 2 s-1 were obtained when nitrocefin was utilized as an optically measurable substrate. Through confocal images and western blot analyses with a specific antibody, the indigenous protein was recognized in S. pneumoniae ATCC 49136. The protein-overexpressed S. pneumonia exhibits a high ampicillin-tolerance ability in vivo. In contrast, the protein-knockout S. pneumonia reveals the ampicillin-sensitive feature relative to the wild type strain. Based on these results, we propose that this protein is a membrane-associated metallo-β-lactamase (MBL) involved in the antibiotic-resistant property of S. pneumoniae. PMID:27214294

  8. Complementary and dose-dependent action of AtCCS52A isoforms in endoreduplication and plant size control.

    PubMed

    Baloban, Mikhail; Vanstraelen, Marleen; Tarayre, Sylvie; Reuzeau, Christophe; Cultrone, Antonietta; Mergaert, Peter; Kondorosi, Eva

    2013-06-01

    · The dimension of organs depends on the number and the size of their component cells. Formation of polyploid cells by endoreduplication cycles is predominantly associated with increases in the cell size and implicated in organ growth. In plants, the CCS52A proteins play a major role in the switch from mitotic to endoreduplication cycles controlling thus the number of mitotic cells and the endoreduplication events in the differentiating cells. · Arabidopsis has two CCS52A isoforms; AtCCS52A1 and AtCCS52A2. Here we focused on their roles in endoreduplication and cell size control during plant development. We demonstrate their complementary and dose-dependent actions that are dependent on their expression patterns. Moreover, the impact of CCS52A overexpression on organ size in transgenic plants was dependent on the expression level; while enhanced expression of the CCS52A genes positively correlated with the ploidy levels, organ sizes were negatively affected by strong overexpression whereas milder overexpression resulted in a significant increase in the organ sizes. · Taken together, these finding support both complementary and dose-dependent actions for the Arabidopsis CCS52A isoforms in plant development and demonstrate that elevated ectopic CCS52A expression positively correlates with organ size, opening a route to higher biomass production.

  9. Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142

    NASA Technical Reports Server (NTRS)

    Schneegurt, M. A.; Sherman, D. M.; Nayar, S.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

    1994-01-01

    It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.

  10. Gene Cluster Involved in the Biosynthesis of Griseobactin, a Catechol-Peptide Siderophore of Streptomyces sp. ATCC 700974▿

    PubMed Central

    Patzer, Silke I.; Braun, Volkmar

    2010-01-01

    The main siderophores produced by streptomycetes are desferrioxamines. Here we show that Streptomyces sp. ATCC 700974 and several Streptomyces griseus strains, in addition, synthesize a hitherto unknown siderophore with a catechol-peptide structure, named griseobactin. The production is repressed by iron. We sequenced a 26-kb DNA region comprising a siderophore biosynthetic gene cluster encoding proteins similar to DhbABCEFG, which are involved in the biosynthesis of 2,3-dihydroxybenzoate (DHBA) and in the incorporation of DHBA into siderophores via a nonribosomal peptide synthetase. Adjacent to the biosynthesis genes are genes that encode proteins for the secretion, uptake, and degradation of siderophores. To correlate the gene cluster with griseobactin synthesis, the dhb genes in ATCC 700974 were disrupted. The resulting mutants no longer synthesized DHBA and griseobactin; production of both was restored by complementation with the dhb genes. Heterologous expression of the dhb genes or of the entire griseobactin biosynthesis gene cluster in the catechol-negative strain Streptomyces lividans TK23 resulted in the synthesis and secretion of DHBA or griseobactin, respectively, suggesting that these genes are sufficient for DHBA and griseobactin biosynthesis. Griseobactin was purified and characterized; its structure is consistent with a cyclic and, to a lesser extent, linear form of the trimeric ester of 2,3-dihydroxybenzoyl-arginyl-threonine complexed with aluminum under iron-limiting conditions. This is the first report identifying the gene cluster for the biosynthesis of DHBA and a catechol siderophore in Streptomyces. PMID:19915026

  11. Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142.

    PubMed

    Bernstein, Hans C; Charania, Moiz A; McClure, Ryan S; Sadler, Natalie C; Melnicki, Matthew R; Hill, Eric A; Markillie, Lye Meng; Nicora, Carrie D; Wright, Aaron T; Romine, Margaret F; Beliaev, Alexander S

    2015-01-01

    To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process. PMID:26525576

  12. Multi-omic dynamics associate oxygenic photosynthesis with nitrogenase-mediated H2 production in Cyanothece sp. ATCC 51142

    DOE PAGES

    Bernstein, Hans C.; Charania, Moiz A.; McClure, Ryan S.; Sadler, Natalie C.; Melnicki, Matthew R.; Hill, Eric A.; Markillie, Lye Meng; Nicora, Carrie D.; Wright, Aaron T.; Romine, Margaret F.; et al

    2015-11-03

    This study combines transcriptomic and proteomic profiling to provide new insights on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in the model cyanobacterium, Cyanothece sp. ATCC 51142. To date, the proposed mechanisms used to describe the energy metabolism processes that support H2 production in Cyanothece 51142 have assumed that ATP and reductant requirements are derived solely from glycogen oxidation and/or cyclic-electron flow around photosystem I. The results from this study present and test an alternative hypothesis by showing that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized withmore » nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the likely role of photocatalytic H2O oxidation as a major participating process.« less

  13. Complete genome sequence of Piscirickettsia salmonis LF-89 (ATCC VR-1361) a major pathogen of farmed salmonid fish.

    PubMed

    Pulgar, Rodrigo; Travisany, Dante; Zuñiga, Alejandro; Maass, Alejandro; Cambiazo, Verónica

    2015-10-20

    Piscirickettsia salmonis, the causative agent of salmonid rickettsial septicemia (SRS), is a significant threat to the healthy and sustainable production of salmonid farming industry. This Gram-negative bacterium, originally isolated from a coho salmon in Southern Chile, produces a systemic infection characterized by colonization of several fish organs. P. salmonis is able to infect, survive, and replicate inside salmonid macrophages however little is known about its mechanisms of pathogenesis. Here, we present the whole genome sequence and annotation of the P. salmonis reference strain LF-89 (ATCC VR-1361). The genome contains one circular chromosome of 3,184,851 bp and three plasmids, pPSLF89-1 (180,124 bp), pPSLF89-2 (33,516 bp) and pPSLF89-3 (51,573 bp). A total of 2850 protein-coding genes, 56 tRNAs and six copies of 5S-16S-23S rRNA. PMID:26220311

  14. Supporting data for comparative proteomic analysis of Listeria monocytogenes ATCC 7644 exposed to a sublethal concentration of nisin

    PubMed Central

    Miyamoto, Kendi Nishino; Mariante Monteiro, Karina; da Silva Caumo, Karin; Rodrigues Lorenzatto, Karina; Bunselmeyer Ferreira, Henrique; Brandelli, Adriano

    2015-01-01

    Here we provide the LC–MS/MS data from a comparative analysis of Listeria monocytogenes ATCC 7644 treated and non-treated with a sublethal concentration of nisin (10−3 mg/mL). Protein samples were analyzed by multidimensional protein identification technology (MudPIT) approach, in an off-line configuration. The raw MS/MS data allowed the detection of 49,591 spectra which resulted in 576 protein identifications. After Scaffold validation, 179 proteins were identified with high confidence. A label-free quantitative analysis based of normalized spectral abundance factor (NSAF) was used and 13 proteins were found differentially expressed between nisin-treated and non-treated cells. Gene ontology analysis of differentially expressed proteins revealed that most of them are correlated to metabolic process, oxidative stress response mechanisms and molecular binding. A detailed analysis and discussion of these data may be found in Miyamoto et al. [1]. PMID:26217729

  15. Enhancing Cellulase Production in Thermophilic Fungus Myceliophthora thermophila ATCC42464 by RNA Interference of cre1 Gene Expression.

    PubMed

    Yang, Fan; Gong, Yanfen; Liu, Gang; Zhao, Shengming; Wang, Juan

    2015-07-01

    The role of CRE1 in a thermophilic fungus, Myceliophthora thermophila ATCC42464, was studied using RNA interference. In the cre1-silenced strain C88, the filter paper hydrolyzing activity and β-1,4-endoglucanase activity were 3.76-, and 1.31-fold higher, respectively, than those in the parental strain when the strains were cultured in inducing medium for 6 days. The activities of β-1,4-exoglucanase and cellobiase were 2.64-, and 5.59-fold higher, respectively, than those in the parental strain when the strains were cultured for 5 days. Quantitative reverse-transcription polymerase chain reaction showed that the gene expression of egl3, cbh1, and cbh2 was significantly increased in transformant C88 compared with the wild-type strain. Therefore, our findings suggest the feasibility of improving cellulase production by modifying the regulator expression, and an attractive approach to increasing the total cellulase productivity in thermophilic fungi.

  16. High quality permanent draft genome sequence of Phaseolibacter flectens ATCC 12775T, a plant pathogen of French bean pods

    DOE PAGES

    Aizenberg-Gershtein, Yana; Izhaki, Ido; Lapidus, Alla; Copeland, Alex; Reddy, TBK; Huntemann, Marcel; Pillay, Manoj; Markowitz, Victor; Göker, Markus; Woyke, Tanja; et al

    2016-01-13

    We report that the Phaseolibacter flectens strain ATCC 12775T (Halpern et al., Int J Syst Evol Microbiol 63:268–273, 2013) is a Gram-negative, rod shaped, motile, aerobic, chemoorganotroph bacterium. Ph. flectens is as a plant-pathogenic bacterium on pods of French bean and was first identified by Johnson (1956) as Pseudomonas flectens. After its phylogenetic position was reexamined, Pseudomonas flectens was transferred to the family Enterobacteriaceae as Phaseolibacter flectens gen. nov., comb. nov. Here we describe the features of this organism, together with the draft genome sequence and annotation. The DNA GC content is 44.34 mol%. The chromosome length is 2,748,442 bp.more » It encodes 2,437 proteins and 89 RNA genes. Ph. flectens genome is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes study.« less

  17. Insights into the fluoride-resistant regulation mechanism of Acidithiobacillus ferrooxidans ATCC 23270 based on whole genome microarrays.

    PubMed

    Ma, Liyuan; Li, Qian; Shen, Li; Feng, Xue; Xiao, Yunhua; Tao, Jiemeng; Liang, Yili; Yin, Huaqun; Liu, Xueduan

    2016-10-01

    Acidophilic microorganisms involved in uranium bioleaching are usually suppressed by dissolved fluoride ions, eventually leading to reduced leaching efficiency. However, little is known about the regulation mechanisms of microbial resistance to fluoride. In this study, the resistance of Acidithiobacillus ferrooxidans ATCC 23270 to fluoride was investigated by detecting bacterial growth fluctuations and ferrous or sulfur oxidation. To explore the regulation mechanism, a whole genome microarray was used to profile the genome-wide expression. The fluoride tolerance of A. ferrooxidans cultured in the presence of FeSO4 was better than that cultured with the S(0) substrate. The differentially expressed gene categories closely related to fluoride tolerance included those involved in energy metabolism, cellular processes, protein synthesis, transport, the cell envelope, and binding proteins. This study highlights that the cellular ferrous oxidation ability was enhanced at the lower fluoride concentrations. An overview of the cellular regulation mechanisms of extremophiles to fluoride resistance is discussed. PMID:27519020

  18. [Effect of cinnamon and lavender oils on FtsZ gene expression in the Staphylococus aureus ATCC 29213].

    PubMed

    Herman, A; Bochenek, J; Herman, A P

    2013-01-01

    This study was designed to determine the effect of lavender and cinnamon oils on FtsZ gene expression in Staphylococcus aureus ATCC 29213. The cinnamon and lavender oils at least partially results from the inhibition of FtsZ transcription and disruption of cell division process at the level of the septum synthesis, what is similar to mechanisms of drug action used in anti-staphylococcal therapies. The presented results could be an important background for the further detailed research, which is needed to clarify the effect of essential oils on FtsZ synthesis at the posttranscriptional level and other stages of cell division process of S. aureus and other pathogenic bacteria.

  19. A role for the diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, in nitrogen cycling for CELSS applications.

    PubMed

    Schneegurt, M A; Sherman, L A

    1996-01-01

    Simple calculations show that fixed nitrogen regeneration in a CELSS may not be as efficient as stowage and resupply of fixed nitrogen compounds. However, fixed nitrogen regeneration may be important for the sustainability and safety of a deployed CELSS. Cyanothece sp. strain ATCC 51142, a unicellular, aerobic, diazotrophic cyanobacterium, with high growth rates and a robust metabolism, is a reasonable candidate organism for a biological, fixed nitrogen regeneration system. In addition, Cyanothece sp. cultures may be used to balance gas exchange ratio imparities between plants and humans. The regeneration of fixed nitrogen compounds by cyanobacterial cultures was examined in the context of a broad computer model/simulation (called CELSS-3D). When cyanothece sp. cultures were used to balance gas exchange imparities, the biomass harvested could supply as much as half of the total fixed nitrogen needed for plant biomass production.

  20. Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142.

    PubMed

    Bernstein, Hans C; Charania, Moiz A; McClure, Ryan S; Sadler, Natalie C; Melnicki, Matthew R; Hill, Eric A; Markillie, Lye Meng; Nicora, Carrie D; Wright, Aaron T; Romine, Margaret F; Beliaev, Alexander S

    2015-11-03

    To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process.

  1. Factors affecting the photoproduction of ammonia from dinitrogen and water by the cyanobacterium Anabaena sp. strain ATCC 33047

    SciTech Connect

    Ramos, J.L.; Guerrero, M.G.; Losada, M.

    1987-04-01

    Synthesis of ammonia from dinitrogen and water by suspensions of Anabaena sp. strain ATCC 33047 treated with the glutamine synthetase inhibitor L-methionine-D,L-sulfoximine is strictly dependent on light. Under otherwise optimal conditions, the yield of ammonia production is influenced by irradiance, as well as by the density, depth, and turbulence of the cell suspension. The interaction among these factors seems to determine the actual amount of light available to each single cell or filament in the suspension for the photoproduction process. Under convenient illumination, the limiting factor in the synthesis of ammonia seems to be the cellular nitrogenase activity level, but under limiting light conditions the limiting factor could, however, be the assimilatory power required for nitrogen fixation. Photosynthetic ammonia production from atmospheric nitrogen and water can operate with an efficiency of ca. 10% of its theoretical maximum, representing a remarkable process for the conversion of light energy into chemical energy.

  2. Control of nitrogenase recovery from oxygen inactivation by ammonia in the cyanobacterium anabaena sp. strain CA (ATCC 33047)

    SciTech Connect

    Smith, R.L.; Van Baalen, C. ); Tabita, F.R. Ohio State Univ., Columbus )

    1990-05-01

    The control of nitrogenase recovery from inactivation by oxygen was studied in Anabaena sp. strain CA (ATCC 33047). Nitrogenase activity (acetylene reduction) in cultures grown in 1% CO{sub 2} in air was inhibited by exposure to 1% CO{sub 2}-99% O{sub 2} and allowed to recover in the presence of high oxygen tensions. Cultures exposed to hyperbaric levels of oxygen in the presence of 10 mM NH{sub 4}NO{sub 3} were incapable of regaining nitrogenase activity, whereas control cultures returned to 65 to 80% of their original activity within about 3 h after exposure to high oxygen tension. In contrast to the regulation of heterocyst differentiation and nitrogenase synthesis, recovery from oxygen inactivation in this organism was shown to be under the control of NH{sub 4}{sup +} rather than NO{sub 3}{sup {minus}}.

  3. Cloning and sequencing of the trpE gene from Arthrobacter globiformis ATCC 8010 and several related subsurface Arthrobacter isolates

    SciTech Connect

    Chernova, T.; Viswanathan, V.K.; Austria, N.; Nichols, B.P.

    1998-09-01

    Tryptophan dependent mutants of Arthrobacter globiformis ATCC 8010 were isolated and trp genes were cloned by complementation and marker rescue of the auxotrophic strains. Rescue studies and preliminary sequence analysis reveal that at least the genes trpE, trpC, and trpB are clustered together in this organism. In addition, sequence analysis of the entire trpE gene, which encodes component I of anthranilate synthase, is described. Segments of the trpE gene from 17 subsurface isolates of Arthrobacter sp. were amplified by PCR and sequenced. The partial trpE sequences from the various strains were aligned and subjected to phylogenetic analysis. The data suggest that in addition to single base changes, recombination and genetic exchange play a major role in the evolution of the Arthrobacter genome.

  4. Control of nitrogenase recovery from oxygen inactivation by ammonia in the cyanobacterium Anabaena sp. strain CA (ATCC 33047).

    PubMed Central

    Smith, R L; Van Baalen, C; Tabita, F R

    1990-01-01

    The control of nitrogenase recovery from inactivation by oxygen was studied in Anabaena sp. strain CA (ATCC 33047). Nitrogenase activity (acetylene reduction) in cultures grown in 1% CO2 in air was inhibited by exposure to 1% CO2-99% O2 and allowed to recover in the presence of high oxygen tensions. Cultures exposed to hyperbaric levels of oxygen in the presence of 10 mM NH4NO3 were incapable of regaining nitrogenase activity, whereas control cultures returned to 65 to 80% of their original activity within about 3 h after exposure to high oxygen tension. In contrast to the regulation of heterocyst differentiation and nitrogenase synthesis, recovery from oxygen inactivation in this organism was shown to be under the control of NH4+ rather than NO3-. PMID:2110151

  5. Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952.

    PubMed

    Ghimire, Gopal Prasad; Oh, Tae-Jin; Liou, Kwangkyoung; Sohng, Jae Kyung

    2008-10-31

    We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA). PMID:18612244

  6. Cloning, expression, and catabolite repression of a gene encoding beta-galactosidase of Bacillus megaterium ATCC 14581.

    PubMed

    Shaw, G C; Kao, H S; Chiou, C Y

    1998-09-01

    A gene encoding beta-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581. Chromosomal beta-galactosidase production could be dramatically induced by lactose but not by isopropyl-beta-D-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of mbgA in the B. megaterium chromosome resulted in loss of lactose-inducible beta-galactosidase production. A 27-bp inverted repeat was found to overlap the mbgA promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s). PMID:9721318

  7. Cloning, Expression, and Catabolite Repression of a Gene Encoding β-Galactosidase of Bacillus megaterium ATCC 14581

    PubMed Central

    Shaw, Gwo-Chyuan; Kao, Hsun-Sheng; Chiou, Chih-Yung

    1998-01-01

    A gene encoding β-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581. Chromosomal β-galactosidase production could be dramatically induced by lactose but not by isopropyl-β-d-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of mbgA in the B. megaterium chromosome resulted in loss of lactose-inducible β-galactosidase production. A 27-bp inverted repeat was found to overlap the mbgA promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s). PMID:9721318

  8. Conditions required for citrate utilization during growth of Lactobacillus casei ATCC334 in chemically defined medium and cheddar cheese extract.

    PubMed

    Díaz-Muñiz, Ilenys; Steele, James L

    2006-10-01

    Conditions required for citrate utilization by Lactobacillus casei ATCC334 were identified. Citrate was utilized by this microorganism in modified Chemically Defined Media (mCDM) as an energy source, solely in the presence of limiting concentrations of galactose. The presence of glucose inhibited citrate utilization by this microorganism even when added in limiting concentrations. Utilization of citrate occurred at pH 6.0 +/- 0.2 and 5.1 +/- 0.2. Together these observations suggest that citrate is an energy source for L. casei in ripening cheese only when the residual levels of carbohydrate post-fermentation are limiting (<2.5 mM), and lactose or glucose are absent. However, citrate utilization by this organism was observed in Cheddar cheese extract (CCE), which naturally contains both lactose and galactose, at the beginning of late-logarithmic phase and regardless of the galactose concentration present in the media.

  9. Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142

    PubMed Central

    Bernstein, Hans C.; Charania, Moiz A.; McClure, Ryan S.; Sadler, Natalie C.; Melnicki, Matthew R.; Hill, Eric A.; Markillie, Lye Meng; Nicora, Carrie D.; Wright, Aaron T.; Romine, Margaret F.; Beliaev, Alexander S.

    2015-01-01

    To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process. PMID:26525576

  10. AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

    PubMed

    McManus, John B; Deng, Ying; Nagachar, Nivedita; Kao, Teh-hui; Tien, Ming

    2016-01-01

    The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx. PMID:26672449

  11. Erythromycin and azithromycin transport into Haemophilus influenzae ATCC 19418 under conditions of depressed proton motive force (delta mu H)

    SciTech Connect

    Capobianco, J.O.; Goldman, R.C. )

    1990-09-01

    The effect of collapsing the electrochemical proton gradient (delta mu H) on ({sup 3}H)erythromycin and ({sup 14}C)azithromycin transport in Haemophilus influenzae ATCC 19418 was studied. The proton gradient and membrane potential were determined from the distribution of (2-{sup 14}C)dimethadione and rubidium-86, respectively. delta mu H was reduced from 124 to 3 mV in EDTA-valinomycin-treated cells at 22{degrees}C with 150 mM KCl and 0.1 mM carbonyl cyanide m-chlorophenylhydrazone. During the collapse of delta mu H, macrolide uptake increased. Erythromycin efflux studies strongly suggested that this increase was not due to an energy-dependent efflux pump but was likely due to increased outer membrane permeability. These data indicated that macrolide entry was not a delta mu H-driven active transport process but rather a passive diffusion process.

  12. Influence of mixotrophic growth on rhythmic oscillations in expression of metabolic pathways in diazotrophic cyanobacterium Cyanothece sp. ATCC 51142.

    PubMed

    Krishnakumar, S; Gaudana, Sandeep B; Digmurti, Madhuri G; Viswanathan, Ganesh A; Chetty, Madhu; Wangikar, Pramod P

    2015-01-01

    This study investigates the influence of mixotrophy on physiology and metabolism by analysis of global gene expression in unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (henceforth Cyanothece 51142). It was found that Cyanothece 51142 continues to oscillate between photosynthesis and respiration in continuous light under mixotrophy with cycle time of ∼ 13 h. Mixotrophy is marked by an extended respiratory phase compared with photoautotrophy. It can be argued that glycerol provides supplementary energy for nitrogen fixation, which is derived primarily from the glycogen reserves during photoautotrophy. The genes of NDH complex, cytochrome c oxidase and ATP synthase are significantly overexpressed in mixotrophy during the day compared to autotrophy with synchronous expression of the bidirectional hydrogenase genes possibly to maintain redox balance. However, nitrogenase complex remains exclusive to nighttime metabolism concomitantly with uptake hydrogenase. This study throws light on interrelations between metabolic pathways with implications in design of hydrogen producer strains.

  13. Optimization of nutrients for gellan gum production by Sphingomonas paucimobilis ATCC-31461 in molasses based medium using response surface methodology.

    PubMed

    Banik, R M; Santhiagu, A; Upadhyay, S N

    2007-03-01

    A molasses based medium for the production of gellan by Sphingomonas paucimobilis ATCC-31461 was developed. Placket-Burman design criterion was applied to study the effect of various nutrient supplements on gellan production using molasses. Among the 20 variables tested, molasses, tryptone, casaminoacid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on gellan production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Placket-Burman design. Most suitable medium composition for production of gellan was (g/l): molasses-112.5; tryptone-1; casaminoacid-1; disodium hydrogen orthophosphate-1; manganese chloride-0.947 and the optimum gellan production was 13.814 g/l.

  14. The Biosynthetic Gene Cluster of Zorbamycin, a Member of the Bleomycin Family of Antitumor Antibiotics, from Streptomyces flavoviridis ATCC 21892

    PubMed Central

    Galm, Ute; Wendt-Pienkowski, Evelyn; Wang, Liyan; George, Nicholas P.; Oh, Tae-Jin; Yi, Fan; Tao, Meifeng; Coughlin, Jane M.; Shen, Ben

    2011-01-01

    The biosynthetic gene cluster for the glycopeptide-derived antitumor antibiotic zorbamycin (ZBM) was cloned by screening a cosmid library of Streptomyces flavoviridis ATCC 21892. Sequence analysis revealed 40 ORFs belonging to the ZBM biosynthetic gene cluster. However, only 23 and 22 ORFs showed striking similarities to the biosynthetic gene clusters for the bleomycins (BLMs) and tallysomycins (TLMs), respectively; the remaining ORFs do not show significant homology to ORFs from the related BLM and TLM clusters. The ZBM gene cluster consists of 16 nonribosomal peptide synthetase (NRPS) genes encoding eight complete NRPS modules, three incomplete didomain NRPS modules, and eight freestanding single NRPS domains or associated enzymes, a polyketide synthase (PKS) gene encoding one PKS module, six sugar biosynthesis genes, as well as genes encoding other biosynthesis and resistance proteins. A genetic system using Escherichia coli-Streptomyces flavoviridis intergeneric conjugation was developed to enable ZBM gene cluster boundary determinations and biosynthetic pathway manipulations. PMID:19081934

  15. AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

    PubMed

    McManus, John B; Deng, Ying; Nagachar, Nivedita; Kao, Teh-hui; Tien, Ming

    2016-01-01

    The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.

  16. Genome sequences of three tunicamycin-producing Streptomyces strains; S. chartreusis NRRL 12338, S. chartreusis NRRL 3882, and S. lysosuperificus ATCC 31396

    Technology Transfer Automated Retrieval System (TEKTRAN)

    S. chartreusis strains NRRL 12338 and NRRL 3882, S. clavuligerus NRRL 3585, and S. lysosuperificus ATCC 31396, are known producers of tunicamycins, and also of charteusins, clavulinate, cephalosporins, holomycins, and calcimycin. Here we announce the sequencing of the S. lysosuperificus and the two...

  17. Draft genome sequence of the extremely acidophilic biomining bacterium Acidithiobacillus thiooxidans ATCC 19377 provides insights into the evolution of the Acidithiobacillus genus.

    PubMed

    Valdes, Jorge; Ossandon, Francisco; Quatrini, Raquel; Dopson, Mark; Holmes, David S

    2011-12-01

    Acidithiobacillus thiooxidans is a mesophilic, extremely acidophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and inorganic sulfur compounds. Here we present the draft genome sequence of A. thiooxidans ATCC 19377, which has allowed the identification of genes for survival and colonization of extremely acidic environments.

  18. Genome sequence of Victivallis vadensis ATCC BAA-548, an anaerobic bacterium from the phylum Lentisphaerae, isolated from the human gastro-intestinal tract

    SciTech Connect

    Van Passel, Mark W.J.; Kant, Ravi; Palva, Airi; Lucas, Susan; Copeland, A; Lapidus, Alla L.; Glavina Del Rio, Tijana; Dalin, Eileen; Tice, Hope; Bruce, David; Goodwin, Lynne A.; Pitluck, Sam; Davenport, Karen W.; Sims, David; Detter, J. Chris; Han, Cliff; Larimer, Frank W; Land, Miriam L; Hauser, Loren John; Kyrpides, Nikos C; Ovchinnikova, Galina; Richardson, Paul; De Vos, Willem M.; Smidt, Hauke; Zoetendal, Erwin G.

    2011-01-01

    Victivallis vadensis ATCC BAA-548 represents the first cultured representative from the novel phylum Lentisphaerae, a deep-branching bacterial lineage. Few cultured bacteria from this phylum are known, and V. vadensis therefore represents an important organism for evolutionary studies. V. vadensis is a strictly anaerobic sugar-fermenting isolate from the human gastro-intestinal tract.

  19. 40 CFR 180.1102 - Trichoderma harzianum KRL-AG2 (ATCC #20847) strain T-22; exemption from requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Trichoderma harzianum KRL-AG2 (ATCC... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1102 Trichoderma harzianum KRL-AG2... of a tolerance is established for residues of the biofungicide Trichoderma harzianum KRL-AG2...

  20. 40 CFR 180.1102 - Trichoderma harzianum KRL-AG2 (ATCC #20847) strain T-22; exemption from requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Trichoderma harzianum KRL-AG2 (ATCC... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1102 Trichoderma harzianum KRL-AG2... of a tolerance is established for residues of the biofungicide Trichoderma harzianum KRL-AG2...