Polar effects of transposon insertion into a minimal bacterial genome
Journal of Bacteriology
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 inserts 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
... stream gene are favored, permitting read-through 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 (_0132) shows a strong preference for Tn5-PuroR insertions transcribed upstream, away from the downstream non-essential gene _0133. This suggested that expression of _0133 due to readthrough from the Tn5-PuroR is lethal when _0132 function is disrupted by transposon insertion. This led to the identification of genes _0133 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 non-essential, and sometimes can give clues to gene function. IMPORTANCE In studies of the minimal genetic requirements for life we used global transposon mutagenesis to identify genes needed for a minimal bacterial genome. Transposon insertion can disrupt function of a gene but can also have polar effects on the expression of adjacent genes. In the Tn5-PuroR construct used in our studies read-through transcription from Tn5-PuroR can drive expression of downstream genes. This results in a preference for Tn5-PuroR insertions transcribed toward a downstream essential or quasi-essential gene within the same operon. Such polar effects can have an impact on the classification of genes as essential, quasi-essential, or non-essential, but this is observed in only a few cases. Also, polar effects of Tn5-PuroR insertion can sometimes give clues to gene function.