Abstract
BackgroundThe soil bacterium Sinorhizobium meliloti and its allies are important nitrogen-fixing bacterial symbionts that cause N2-fixing nodules on the roots of legumes. Chromosomal ß-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.ResultsThe 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.ConclusionsA 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.
Highlights
The soil bacterium Sinorhizobium meliloti and its allies are important nitrogen-fixing bacterial symbionts that cause N2-fixing nodules on the roots of legumes
PVMG was constructed by replacing a BamH I-Xba I fragment at the end of multiple cloning sites (MCS) in pVO155 with the 25-bp BamH I-Xba I synthetic DNA linker containing three translation terminations and a Xho I site
DNA sequencing and double/triple endonuclease digests of the plasmid confirmed that pVMG retains the original promoterless uidA gene, seven of the 8 restriction enzymes in multiple cloning sites (MCS), a E. coli trpA terminator upstream of the MCS to prevent read-through from the vector, a unique Hind III site adjacent to the trpA, a unique Bgl II and the pUC origin of replication
Summary
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 ß-glucuronidase gene (uidA) transcriptional fusions are frequently used to monitor the expression of bacterial genes during the symbiosis. The chromosomal uidA transcriptional fusions are frequently used for monitoring in vivo expression of bacterial genes for at least three reasons: First, the fusions allow the transcriptional activities of bacterial genes to be monitored at their nature levels [1,2,3,4]. The fusions do not need antibiotics for maintaining their stability in the genome. They avoid problems that associate with replicating plasmid systems which can disrupt regulation of expression due to copy number effects [1, 3,4,5]. As higher plants lack ß-glucuronidase activity [6], the uidA gene provides a sensitive enzyme assay for which a broad range of substances are available
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