Polar effects of transposon insertion into a minimal bacterial genome

Abstract

Global transposon mutagenesis is a valuable tool for identifying genes required for cell viability. Here we present a global analysis of the orientation of viable Tn5-Puror (Tn5-puromycin resistance) insertions into the near-minimal bacterial genome of JCVI-syn2.0. Sixteen of the 478 protein-coding genes show a noticeable asymmetry in the orientation of disrupting insertions of Tn5-Puror. Ten of these are located in operons, upstream of essential or quasi-essential genes. Inserts transcribed in the same direction as the downstream gene are favored, permitting readthrough transcription of the essential or quasi-essential gene. Some of these genes were classified as quasi-essential solely because of polar effects on the expression of downstream genes. Three genes showing asymmetry in Tn5-Puror insertion orientation prefer the orientation that avoids collisions between read-through transcription of Tn5-Puror and transcription of an adjacent gene. One gene (JCVISYN20132 [abbreviated here as "0132"]) shows a strong preference for Tn5-Puror insertions transcribed upstream, away from the downstream nonessential gene0133. This suggested that expression of0133 due to read-through from Tn5-Puror is lethal when0132 function is disrupted by transposon insertion. This led to the identification of genes0133 and 0132 as a toxin-antitoxin pair. The three remaining genes show read-through transcription of Tn5-Puror directed downstream and away from sizable upstream intergenic regions (199 bp to 363 bp), for unknown reasons. In summary, polar effects of transposon insertion can, in a few cases, affect the classification of genes as essential, quasi-essential, or nonessential and sometimes can give clues to gene function.