Abstract
BackgroundThe Rhizobiaceae family of Gram-negative bacteria often engage in symbiosis with plants of economic importance. Historically, genetic studies to identify the function of individual genes, and characterize the biology of these bacteria have relied on the use of classical transposon mutagenesis. To increase the rate of scientific discovery in the Rhizobiaceae there is a need to adapt high-throughput genetic screens like insertion sequencing for use in this family of bacteria. Here we describe a Rhizobiaceae compatible MmeI-adapted mariner transposon that can be used with insertion sequencing for high-throughput genetic screening.ResultsThe newly constructed mariner transposon pSAM_Rl mutagenized R. leguminosarum, S. meliloti, and A. tumefaciens at a high frequency. In R. leguminosarum, mutant pools were generated that saturated 88% of potential mariner insertions sites in the genome. Analysis of the R. leguminosarum transposon insertion sequencing data with a previously described hidden Markov model-based method resulted in assignment of the contribution of all annotated genes in the R. leguminosarum 3841 genome for growth on a complex medium. Good concordance was observed between genes observed to be required for growth on the complex medium, and previous studies.ConclusionsThe newly described Rhizobiaceaee compatible mariner transposon insertion sequencing vector pSAM_Rl has been shown to mutagenize at a high frequency and to be an effective tool for use in high-throughput genetic screening. The construction and validation of this transposon insertion sequencing tool for use in the Rhizobiziaceae will provide an opportunity for researchers in the Rhizobiaceae community to use high-throughput genetic screening, allowing for significant increase in the rate of genetic discovery, particularly given the recent release of genome sequences from many Rhizobiaceae strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0298-z) contains supplementary material, which is available to authorized users.
Highlights
The Rhizobiaceae family of Gram-negative bacteria often engage in symbiosis with plants of economic importance
We found that the MmeI-adapted mariner transposon harbored on pSAM_Rl could mutagenize R. leguminosarum, A. tumefaciens, and S. meliloti at a high frequency
This suggests that the combination of mariner based transposon insertion sequencing with the Bayesian based hidden Markov model (HMM) analysis would yield accurate and full genome level results
Summary
The Rhizobiaceae family of Gram-negative bacteria often engage in symbiosis with plants of economic importance. To increase the rate of scientific discovery in the Rhizobiaceae there is a need to adapt high-throughput genetic screens like insertion sequencing for use in this family of bacteria. The Rhizobiaceae is a family of alpha-proteobacteria containing three agriculturally important genera of soil bacteria: Rhizobium, Sinorhizobium and Agrobacterium [25] Members in these genera share a unique relationship with plant hosts. Sinorhizobium are both able to enter into an endosymbiotic mutualism with certain species of leguminous plants, in which the Rhizobia fix atmospheric nitrogen into a biologically available form for the plant in return for fixed carbon and energy [26] This symbiosis is important in the context of agriculturally produced pulse crops, where the Rhizobium legume symbiosis affords farmers the ability to reduce the rate of synthetic nitrogen fertilizers application [27]. The formation of several galls at the stem root interface results in a plant infection known as crown gall, that can have a significant impact on the crop yield of stone fruits, berries, and nuts [29]
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