Tn-seq of Thermus thermophilus genome reveals unexpected tolerance to insertions in bacterial common essential genes.

A large library based on a Tn5 minitransposon carrying a thermostable kanamycin resistance gene was prepared using Thermus thermophilus HB27 genomic DNA as target. To increase the yield of transformants, DNA from the in vitro transposition reaction was amplified using isothermal multiple displacement amplification. The resulting product was first transformed into the high-transformation efficiency{Delta} addAB/ppol strain and then into a wild-type HB27 strain. Tn-seq analysis of the libraries showed that almost all genes contained insertions and the distribution of the number of insertions per gene was unimodal, unlike the bimodal distribution reported in most Tn-seq analyses, thus hindering the discrimination of required or essential genes. Upon comparing the Tn-seq results with gene conservation in pangenomic analysis from Thermus thermophilus to Deiococcota levels, as well as with available HB27 RNA-seq data, we observed a very low correlation between core genes or gene transcription levels and Tn-seq insertion frequency. Notably, many genes largely deemed part of the essential bacterial core, supporting critical cellular pathways, showed relatively high transposon insertion numbers. In the case of DNA repair routes, which are essential but somewhat redundant, our results align well with previously published essentiality data, indicating that many genes are dispensable and permissive to insertions. The analysis of these striking results in the context of Thermus biology suggests that the polyploidy of the Thermus genome and the differential stability of proteins may explain the apparent non-essentiality of key genes. ImportanceThe Tn-seq technique, random insertion of transposons into an organisms genome followed by deep sequencing of the resulting population, has enabled precise identification of genes necessary for that organism to live under specified conditions. We have performed Tn-seq on the extremophilic bacterium Thermus thermophilus to gain information on the genes required for thriving at high temperature. Unexpectedly, we detected transposon insertions in the practical totality of the genes of this organism, hindering a clear discrimination of essential genes. Furthermore, a comparison with core genes derived from a pangenome analysis, spanning from the Thermus genus up to the phylum level, failed to establish a correlation between gene conservation and Tn-seq results. The polyploidy of the genome of this organism, along with potential regulatory mechanisms, may explain why commonly essential bacterial genes tolerate transposon insertions, a characteristic that appears particular for this organism.