|RATHNAIA, GOVARDHAN - University Of Nebraska|
|ZINNIEL, DENISE - University Of Nebraska|
|GROHN, YRJO - Cornell University - New York|
|BARLETTA, RAUL - University Of Nebraska|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/5/2015
Publication Date: 12/4/2015
Citation: Rathnaia, G., Bannantine, J.P., Bayles, D.O., Zinniel, D., Stabel, J.R., Grohn, Y., Barletta, R. 2015. Analysis of Mycobacterium avium subspecies paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies to novel mutant discovery. Meeting Abstract. Rathnaia, G., Bannantine, J.P., Bayles, D., Zinniel, D., Stabel, J.R., Grohn, Y., Barletta, R., 2015. CRWAD Conference.
Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne’s disease, is one of the most important bacterial pathogens in ruminants. The lack of efficacious control measures demands a thorough understanding of MAP pathogenesis to develop new vaccines and diagnostic tests. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental genetic technology utilized to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis was performed to compare the insertion sites for the classical mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99% of all MAP genes. Genome analysis of the MAP K-10 strain showed that 710 (16.3%) of all open reading frames do not possess IS1096 recognition sites, while only 37 (0.85%) do not have the recognition site for MycoMarT7 insertions. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096 derived transposons. Analysis of pools containing 109 MycomarT7 and 108 Tn5367 showed a predilection to insert within intergenic regions, suggesting that MycoMarT7 is more adequate to generate a comprehensive library to analyze gene essentiality under various conditions. However, we also uncovered the novel finding that both transposons have loci-dependent biases with Tn5367 being the most skewed. These loci-dependent transposition biases lead to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Our experiments also define a useful platform for gene discovery by allowing the rapid production of smaller mutant pools that could be readily sequenced and assayed for specific properties. Herein, we demonstrated these features by isolating three novel mutants for each transposon.