Surfactant degradative plasmids

It has been shown that the degradation of anionlc, cationic and ampholytic surfactants by pseudomonads can be controlled by the plasmids with size of 60–130 kb. Most plasmid strains that degrade surfactants are capable of conjugative transfer and elimination from bacteria cells. Restriction patterns of plasmids do not reveal significant homology between plasmids.Показано, что деградация многих анионных, катионных и амфолитных поверхностно-активных веществ у псевдомонад контролируется плазмидами размером 60–130 тыс. п. н. Большинство плазм ид способны к конъюгативному переносу и элиминации из бактериальных клеток. Полученные первичные рестрикционные карты данных плазм ид имеют значительные различия.Показано, що деградація багатьох аніонних, катіонних та амфолітних поверхнево-активних речовин (ПАР) у псевдомонад контролюються плазмідами розміром 60–130 тис. п. н. Більшість плазмід здатні до кон'югативного переносу та елімінації з бактеріальних клітин. Створені первинні рестрикційні карти плазмід біодеградації ПАР мають значну різницю

Drought tolerance in Pinus halepensis seed sources as identified by distinctive physiological and molecular markers

[EN] Drought is one of the main constraints determining forest species growth, survival and productivity, and therefore one of the main limitations for reforestation or afforestation. The aim of this study is to characterize the drought response at the physiological and molecular level of different Pinus halepensis (common name Aleppo pine) seed sources, previously characterized in field trials as drought-sensitive or drought-tolerant. This approach aims to identify different traits capable of predicting the ability of formerly uncharacterized seedlings to cope with drought stress. Gas-exchange, water potential, photosynthetic pigments, soluble sugars, free amino acids, glutathione and proteomic analyses were carried out on control and drought-stressed seedlings in greenhouse conditions. Gas-exchange determinations were also assessed in field-planted seedlings in order to validate the greenhouse experimental conditions. Drought-tolerant seed sources presented higher values of photosynthetic rates, water use efficiency, photosynthetic pigments and soluble carbohydrates concentrations. We observed the same pattern of variation of photosynthesis rate and maximal efficiency of PSII in field. Interestingly drought-tolerant seed sources exhibited increased levels of glutathione, methionine and cysteine. The proteomic profile of drought tolerant seedlings identified two heat shock proteins and an enzyme related to methionine biosynthesis that were not present in drought sensitive seedlings, pointing to the synthesis of sulfur amino acids as a limiting factor for drought tolerance in Pinus halepensis. Our results established physiological and molecular traits useful as distinctive markers to predict drought tolerance in Pinus halepensis provenances that could be reliably used in reforestation programs in drought prone areas.This study is a part of the research project: "Application of molecular biology techniques in forest restoration in Mediterranean environments, PAID-05-11" funded by the Universitat Politecnica de Valencia (UPV), program for supporting R&D of new multidisciplinary research lines. The authors are grateful to the Ministerio de Economia y Competitividad AGL2014-57431-P. AV was supported by project Survive-2 (CGL2015-69773-C2-2-P MINECO/FEDER) by the Spanish Government and Prometeo program (DESESTRES-Generalitat Valenciana). CEAM is funded by Generalitat Valenciana.Taïbi, K.; Campo García, ADD.; Vilagrosa, A.; Belles Albert, JM.; López-Gresa, MP.; Pla, D.; Calvete Chornet, JJ.... (2017). 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Auxotrophic Selection Strategy for Improved Production of Coenzyme B-12 in Escherichia coli

The production of coenzyme B-12 using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B-12 in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B-12 using a previously engineered coenzyme B-12-producing E. coli strain. To select a high producer, the coenzyme B-12-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B-12-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B-12 validated the coenzyme B-12-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B-12 production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B-12-producing strains

Stabilized MetA decreases the frequencies of persisters in different <i>E. coli</i> strains at elevated temperature.

<p>Cells of the strains W3110 and W3110-LYD (A), WErph⁺ and WErph⁺-LYD (B), grown overnight for 16 h in M9 glucose medium at 37 or 42°C, were diluted to an OD₆₀₀ of 0.1 in fresh M9 glucose medium supplemented with ampicillin and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a>.</p

Stabilization of Homoserine-O-Succinyltransferase (MetA) Decreases the Frequency of Persisters in <i>Escherichia coli</i> under Stressful Conditions

<div><p>Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions (heat, oxidative, acidic stress) induces the accumulation of aggregated proteins and enhances formation of persister cells in <i>Escherichia coli</i> cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-<i>o</i>-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in <i>E. coli</i> growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native <i>metA</i> gene on the <i>E. coli</i> K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in <i>E. coli</i> K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.</p></div

Influence of stabilized MetA protein on the <i>E.coli</i> WE strain growth under stressful conditions.

<p>The WE and WE-LYD strains were incubated in M9 glucose medium at 44°C for 10 h (A) or in M9 glucose medium (pH 6.0) supplemented with 20 mM sodium acetate at 37°C for 28 h (B) in an automatic growth-measuring incubator. The average of two independent experiments is presented.</p

Dependence of persister formation on stabilized MetA protein.

<p>Overnight cultures of the strains WE and WE-LYD grown for 16 h in M9 glucose medium at 37 or 42°C were diluted to an OD₆₀₀ of 0.1 in fresh M9 glucose medium supplemented with ampicillin (A) or ofloxacin (B) and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a>. Soluble and insoluble protein fractions were purified from the cultures grown in M9 glucose medium at 37 or 42°C to an OD₆₀₀ = 1.0, subjected to 12% SDS-PAGE followed by Western blotting using rabbit anti-MetA antibody (C). The MetA in the samples was quantified through densitometry using WCIF ImageJ software. The MetA amount from the WE cells grown at 37°C was set to 1 (D). The data are presented as the average of two independent experiments.</p

Effect of L-methionine on the frequency of persisters at different temperatures.

<p>The 16-h cultures of the strains WE (A, B) and JW0195 (C) grown in M9 glucose medium with or without L-methionine (50 µg/ml) at 37 or 42°C were diluted to an OD₆₀₀ of 0.1 in fresh M9 glucose medium supplemented with ampicillin (A, C) or ofloxacin (B) and incubated at 37°C for 10 hours. Samples were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110504#s2" target="_blank">Materials and Methods</a>.</p

Surfactant degradative plasmids

It has been shown that the degradation of anionlc, cationic and ampholytic surfactants by pseudomonads can be controlled by the plasmids with size of 60–130 kb. Most plasmid strains that degrade surfactants are capable of conjugative transfer and elimination from bacteria cells. Restriction patterns of plasmids do not reveal significant homology between plasmids.Показано, что деградация многих анионных, катионных и амфолитных поверхностно-активных веществ у псевдомонад контролируется плазмидами размером 60–130 тыс. п. н. Большинство плазм ид способны к конъюгативному переносу и элиминации из бактериальных клеток. Полученные первичные рестрикционные карты данных плазм ид имеют значительные различия.Показано, що деградація багатьох аніонних, катіонних та амфолітних поверхнево-активних речовин (ПАР) у псевдомонад контролюються плазмідами розміром 60–130 тис. п. н. Більшість плазмід здатні до кон'югативного переносу та елімінації з бактеріальних клітин. Створені первинні рестрикційні карти плазмід біодеградації ПАР мають значну різницю

Strains and plasmids used in this study.

<p>Ap^r, ampicillin resistance; <i>kan</i>, kanamycin resistance gene.</p><p>Strains and plasmids used in this study.</p