RESUMEN
Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.
Asunto(s)
Proteínas Bacterianas/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , NADH NADPH Oxidorreductasas/genética , Fosfitos/metabolismo , Fósforo/metabolismo , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Proteínas Bacterianas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Ingeniería Genética/métodos , Marcadores Genéticos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fosfitos/farmacología , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genética , Selección Genética , Transformación GenéticaRESUMEN
The diversity of bacterial communities of shallow (< or = 100 m depth) oil seep marine sediments from the southern Gulf of Mexico was evaluated. The geochemical properties of seep sediments were characterized as well as their microbial diversity in oil seep and control sediments. Bacteria were identified through molecular tools as belonging to the genera Marinobacter, Idiomarina, Marinobacterium, Frauteria and an unknown bacterium. Bacteria might be important components of microbial communities in Total Petroleum Hydrocarbon (TPH)-containing environments, displaying either facultative metabolism or able to grow only in petroleum-containing media. The identification of bacteria in shallow oil seep sediments could be used as indicators of marine hydrocarbons in southeastern Gulf of Mexico.