RESUMEN
Saanich Inlet (British Columbia, Canada) is a seasonally anoxic fjord characterized by high rates of both methane production and consumption. In this study, the diversity of microbial populations residing in intermediate waters, characterized by having a high methane content, was assessed using CH(4)-microcosm experiments coupled with PCR surveys of phylogenetic (16S rRNA gene) and functional gene markers (pmoA and fhcD genes). The experiments revealed that bacteria represented by sequences affiliated with Methylomicrobium within the Methylococcales, Methylophaga and Cycloclasticus within the Thiotrichales, and uncultured Planctomycetes were enriched in response to CH(4) addition.
Asunto(s)
Bacterias/clasificación , Bacterias/metabolismo , Biota , Metano/metabolismo , Agua de Mar/microbiología , Bacterias/genética , Proteínas Bacterianas/genética , Colombia Británica , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Cell-to-cell heterogeneity in gene expression and growth parameters was assessed in the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methylotrophy promoter (P(mxaF)) and gfp(uv) (encoding a variant of green fluorescent protein [GFPuv]) was used to assess single-cell gene expression. Using a flowthrough culture system and laser scanning microscopy, data on fluorescence and cell size were obtained over time through several growth cycles for cells grown on succinate or methanol. Cells were grown continuously with no discernible lag between divisions, and high cell-to-cell variability was observed for cell size at division (2.5-fold range), division time, and growth rate. When individual cells were followed over multiple division cycles, no direct correlation was observed between the growth rate before a division and the subsequent growth rate or between the cell size at division and the subsequent growth rate. The cell-to-cell variability for GFPuv fluorescence from the P(mxaF) promoter was less, with a range on the order of 1.5-fold. Fluorescence and growth rate were also followed during a carbon shift experiment, in which cells growing on succinate were shifted to methanol. Variability of the response was observed, and the growth rate at the time of the shift from succinate to methanol was a predictor of the response. Higher growth rates at the time of the substrate shift resulted in greater decreases in growth rates immediately after the shift, but full induction of P(mxaF)-gfp(uv) was achieved faster. These results demonstrate that in M. extorquens, physiological heterogeneity at the single-cell level plays an important role in determining the population response to the metabolic shift examined.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Methylobacterium extorquens/crecimiento & desarrollo , Methylobacterium extorquens/genética , División Celular/efectos de los fármacos , División Celular/genética , División Celular/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Metano/farmacología , Methylobacterium extorquens/citología , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Mutación , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Succinatos/farmacología , Factores de TiempoRESUMEN
A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/beta-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/beta-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/beta-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3beta inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3beta inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/beta-catenin signaling is a normal physiological response to load and that activation of the Wnt/beta-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.