Use of Plasmid pVMG to Make Transcriptional ?-Glucuronidase Reporter Gene Fusions in the Rhizobium Genome for Monitoring the Expression of Rhizobial Genes In Vivo

摘要

The soil bacterium Sinorhizobium meliloti and its allies are important nitrogen-fixing bacterial symbionts that cause N2-fixing nodules on the roots of legumes. Chromosomal ?-glucuronidase gene (uidA) transcriptional fusions are frequently used to monitor the expression of bacterial genes during the symbiosis. However, the construction of the fusions is laborious. The narrow-host-range, fusion selective plasmid pVMG was constructed and used as a vector for the construction of chromosomal uidA transcriptional fusions in the S. meliloti genome. Translation termination codons were added in all three reading frames upstream of the promoterless uidA in this vector to ensure transcriptional fusions. pVMG replicated to high copy number in Escherichia coli, offering advantages for the isolation of fusion-containing plasmids and the restriction analysis. Genomic locations of uidA fusions were verified in a simple PCR experiment. All these helps reduce the sample processing time and efforts. As a demonstration of its usefulness, the N-acyl homoserine lactone (AHL) signal synthase gene promoter was fused to uidA and shown to be expressed by S. meliloti in the senescence zone of the nodule on the host plant, M. truncatula. This indicates the presence of AHL signals at the late stages of symbiosis. A simple, pVMG-based method for construction of chromosomal uidA transcriptional fusions has been successfully used in the model rhizobium S. meliloti. It is also applicable for other rhizobial strains.