RESUMO
Sinorhizobium meliloti can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of S. meliloti to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of S. meliloti Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (relA, rpoE2, and hpr) and a DNA repair gene (uvrC). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance, uvrA, uvrB, and uvrC deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that uvr gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance.
Assuntos
Reparo do DNA , Dessecação , Regulação Bacteriana da Expressão Gênica , Viabilidade Microbiana , Sinorhizobium meliloti/fisiologia , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Medicago sativa , Mutagênese Insercional , Análise de Sequência de DNA , Sinorhizobium meliloti/genéticaRESUMO
The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to beta-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput beta-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.
Assuntos
Fusão Gênica Artificial , Proteínas de Bactérias/metabolismo , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Coloração e Rotulagem/métodos , Proteínas de Bactérias/genética , DNA Bacteriano , Perfilação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Glucuronidase/genética , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Análise de Sequência de DNARESUMO
Phytate, the storage form of phosphate in seeds and grains, is a major form of environmental phosphate loading from fertilizer inputs and agricultural runoff. We have investigated the ability of Tetrahymena populations to grow on phytate as their sole phosphate source. Populations grew equally well in chemically defined medium with phosphate and medium in which the phosphate was replaced with phytate in comparable concentrations between 0.5 mM and 6 mM. Intracellular phytate concentrations of cells grown in phytate showed a 4-6-fold increase over those grown in phosphate when measured during the late stage of exponential growth. These results demonstrate that phytate can provide a source of adequate phosphate for sustained growth in phytate-rich environments.
