Naturally occurring DNA transfer system associated with membrane vesicles in cellulolytic Ruminococcus spp. of ruminal origin

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen

New Measure of Insulin Sensitivity Predicts Cardiovascular Disease Better than HOMA Estimated Insulin Resistance

10.1371/journal.pone.0074410PLoS ONE89-POLN

Efeito de aditivos para silagem e duração da exposição aeróbia sobre a degradabilidade in vitro e recuperação da matéria seca de sorgo para silagem em dois estágios de maturação

The effects of silage additives and duration of aerobic exposure on the in vitro degradability and recovery of dry matter (DM) of ensiled forage sorghum in the two maturity stages of 90 and 110 days of growth were evaluated. The forage chopped into 2.5 cm pieces received one of four following treatments: without additive (control), enzyme (0.1% of the fresh matter), bacteria inoculum (106 cfu / g of fresh material) and enzymes together with the inoculum; Then it was placed in the laboratory built with PVC (Polyvinil Chloride) with a capacity of 1.6 kg. Two silos were opened per treatment in each stage of maturity after 100 days of fermentation. The resulting silage was exposed to aerobic conditions in styrofoam containers lined with plastic. After 0, 3, and 7 days of aerobic exposure, the in vitro degradability of the DM and the recovery of the DM were determined. DM degradability was similar for both maturity stages and between additives, however, it decreased (P &lt;.05) after 7 days (54.2%) of aerobic exposure when compared to 0 (57.5%) and 3 days (56.1 %). The DM recovery was lower (P &lt;.05) in silage sorghum at 90 days of growth with the 3 additives (87.2 to 89.8%) than without these (95.9%), while in silage sorghum at 110 days it was lower with the bacterial inoculum than without it (89.5 and 91.6% vs. 91.3 and 94.7%). It is concluded that the addition of the additives did not improve the aerobic stability of the forage sorghum silage and that the deterioration of the silage is progressive with the exposure time.Se evaluaron los efectos de aditivos para ensilar y duración de exposición aeróbica sobre la degradabilidad in vitro y recuperación de la materia seca (MS) de sorgo forrajero ensilado en los dos estados de madurez de 90 y 110 días de crecimiento. El forraje picado en pedazos de 2.5 cm recibió uno de cuatro tratamientos siguientes: sin aditivo (control), enzima (0.1% de la materia fresca), inóculo de bacterias (106 ufc/g de material fresco) y enzimas junto al inóculo; luego se situó de laboratorio construidos con PVC (Polyvinil Chloride) de capacidad 1.6 kg. Se abrieron dos silos por tratamiento en cada estado de madurez después de 100 días de fermentación. El ensilaje resultante fue expuesto a condiciones aeróbicas en envases de “styrofoam” forrados de plástico. Después de 0, 3, y 7 días de exposición aeróbica se determinó la degradabilidad in vitro de la MS y la recuperación de la MS. La degradabilidad de la MS fue similar para ambos estados de madurez y entre aditivos, sin embargo, disminuyó (P&lt;.05) luego de 7 días (54.2%) de exposición aeróbica al compararse con 0 (57.5%) y 3 días (56.1%). La recuperación de la MS fue menor (P&lt;.05) en sorgo ensilado a 90 días de crecimiento con los 3 aditivos (87.2 a 89.8%) que sin estos (95.9%), mientras en sorgo ensilado a 110 días fue menor con el inóculo bacteriano que sin éste (89.5 y 91.6% vs. 91.3 y 94.7%). Se concluye que la adición de los aditivos no mejoró la estabilidad aeróbica del ensilaje de sorgo forrajero y que el deterioro del ensilaje es progresivo con el tiempo de exposición.Avaliaram-se os efeitos dos aditivos para silagem e da duração da exposição aeróbia na degradabilidade in vitro e recuperação da matéria seca (MS) do sorgo forrageiro ensilado nos dois estádios de maturação de 90 e 110 dias de crescimento. A forragem picada em pedaços de 2,5 cm recebeu um dos quatro seguintes tratamentos: sem aditivo (controle), enzima (0,1% da matéria fresca), inóculo de bactéria (106 ufc / g de matéria fresca) e enzimas junto com o inóculo; Em seguida foi colocado em laboratório construído em PVC (Policloreto de Vinila) com capacidade para 1,6 kg. Dois silos foram abertos por tratamento em cada estágio de maturação após 100 dias de fermentação. A silagem resultante foi exposta a condições aeróbicas em recipientes de isopor revestidos de plástico. Após 0, 3 e 7 dias de exposição aeróbia, a degradabilidade in vitro da MS e a recuperação da MS foram determinadas. A degradabilidade da MS foi semelhante para ambos os estágios de maturação e entre os aditivos, porém diminuiu (P &lt;0,05) após 7 dias (54,2%) de exposição aeróbia quando comparada a 0 (57,5%) e 3 dias (56,1%). A recuperação de MS foi menor (P &lt;0,05) na silagem de sorgo aos 90 dias de crescimento com os 3 aditivos (87,2 a 89,8%) do que sem estes (95,9%), enquanto na silagem de sorgo aos 110 dias foi menor com a bacteriana inóculo do que sem ele (89,5 e 91,6% vs. 91,3 e 94,7%). Conclui-se que a adição dos aditivos não melhorou a estabilidade aeróbia da silagem de sorgo forrageiro e que a deterioração da silagem é progressiva com o tempo de exposição

Naturally Occurring DNA Transfer System Associated with Membrane Vesicles in Cellulolytic Ruminococcus spp. of Ruminal Origin

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen