Dataset Information

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

ABSTRACT: Background: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. However, the construction of the fusions is laborious. Results: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. Conclusions: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.

SUBMITTER: Gao M

PROVIDER: S-EPMC6498626 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Use of Plasmid pVMG to Make Transcriptional ß-Glucuronidase Reporter Gene Fusions in the <i>Rhizobium</i> Genome for Monitoring the Expression of Rhizobial Genes In Vivo.

Gao Mengsheng M Benge Anne A Wu Tai-Jung TJ Javier Regina R

Biological procedures online 20190503

<h4>Background</h4>The soil bacterium <i>Sinorhizobium meliloti</i> and its allies are important nitrogen-fixing bacterial symbionts that cause N<sub>2</sub>-fixing nodules on the roots of legumes. Chromosomal ß-glucuronidase gene (<i>uidA)</i> transcriptional fusions are frequently used to monitor the expression of bacterial genes during the symbiosis. However, the construction of the fusions is laborious.<h4>Results</h4>The narrow-host-range, fusion selective plasmid pVMG was constructed and u ...[more]

PMID: 31073281

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Project description:BackgroundA traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of R. etli CFN42 predicts the presence of panC and panB genes clustered together on the 642 kb plasmid p42f and a second copy of panB on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the Rhizobiales with multipartite genomes, and assess the possibility that the panCB genes may have reached plasmids by horizontal gene transfer.ResultsAnalysis of mutants confirmed that the panC and panB genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of panCB genes among members of the Rhizobiales showed that only R. etli and R. leguminosarum strains carry panCB genes in plasmids. The panCB phylogeny attested a common origin for chromosomal and plasmid-borne panCB sequences, suggesting that the R. etli and R. leguminosarum panCB genes are orthologs rather than xenologs. The panCB genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium.ConclusionsThis study shows experimental evidence that core panCB genes located in plasmids of R. etli and R. leguminosarum are indispensable for the synthesis of pantothenate. The unusual presence of panCB genes in plasmids of Rhizobiales may be due to an intragenomic transfer from chromosome to plasmid. Plasmid p42f encodes other functions required for growth in minimal medium. Our results support the hypothesis of cooperation among different replicons for basic cellular functions in multipartite rhizobia genomes.

Dataset Information

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

Publications

Use of Plasmid pVMG to Make Transcriptional ß-Glucuronidase Reporter Gene Fusions in the <i>Rhizobium</i> Genome for Monitoring the Expression of Rhizobial Genes In Vivo.

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