Construction of a mariner -based transposon vector for use in insertion sequence mutagenesis in selected members of the Rhizobiaceae

Perry and Yost BMC Microbiology 2014, 14:298 http://www.biomedcentral.com/1471-2180/14/298 METHODOLOGY ARTICLE Open Access Construction of a mariner-based transposon vector for use in insertion sequence mutagenesis in selected members of the Rhizobiaceae Benjamin J Perry and Christopher K Yost* Abstract Background: The 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. Results: The 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. Conclusions: The 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. Background Insertion sequencing (INSeq) is a technique for high throughput forward genetic screening that has recently become a favorable approach to studying gene function at the genome scale [1,2]. INSeq relies on the use of next-generation DNA sequencing to audit the presence of hundreds of thousands of unique transposon insertions present in a pool of mutants that collectively saturate that organism’s genome with transposition events [3-6]. In general, INSeq based methods can use two different methods to analyze gene function. The first relies on sequencing the transposon insertions sites in an * Correspondence: Department of Biology, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada input pool and an output pool of transposon mutants, and using the differential representation of mutants in each pool to infer the functional role of each gene with sufficient representation of insertion sites [7]. The second method relies on creating a mutant pool sufficiently large and complex that it saturates the genome and allows for analysis of regions with statistically fewer, or no, insertions than expected using a non-parametric [8], Bayesian model [9], or hidden Markov model (HMM) based analysis [10,11]. Both approaches have been applied to several species of bacteria to investigate genes involved in colonization of hosts [12-14], resistance to antibiotics [15], characterizing metabolic pathways [16,17], deducing core essential genomes [18-24], and recently, examining genes involved in colonizing soil environments [7]. © 2014 Perry and Yost; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perry and Yost BMC Microbiology 2014, 14:298 http://www.biomedcentral.com/1471-2180/14/298 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. Rhizobium, and 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 particularly 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]. Conversely, the relationship of Agrobacterium with its plant host is parasitic. In this symbiosis, Agrobacterium infects the tissues of a plant host and transforms specific virulence genes into the host’s DNA, resulting in tumorgenic growth with altered cellular metabolism that the bacteria then colonize [28]. 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]. Genetic research in Rhizobium, Sinorhizobium, and Agrobacterium has relied heavily on the use of transposon mutagenesis screens. Perhaps the most commonly used transposon in the Rhizobiaceae is the Tn5 transposon [30-32]. The use of Tn5 genetic screens is numerous and has helped to elucidate genes involved in metabolism [33-35], desiccation tolerance [36,37], and cell envelope physiology [38] for example. Implementation of transposon mutagenesis with the high-throughput techniques of INSeq promise to accelerate the rate at which genetic research in the Rhizobiaceae is currently performed. Furthermore, it would allow for comprehensive genome screens for genes involved in host interactions, metabolism, survival, and possibly plasmid maintenance, under any testable condition. The mariner class of transposon is a host independent transposon that unlike the random insertion transposons such as Tn5 is known to specifically insert into an organism's genome at thymine-adenine (‘TA’) motifs [39]. Because of this defined insertion preference, transposition events can be modeled in silico in any sequenced genome to understand the defined number of insertion locations that exist. This type of analysis can be further refined to examine insertions per gene or within any defined region of interest in the genome. Furthermore, using a transposon with (...truncated)