Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants

Abstract

Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

“…Transposon insertion sequencing (Tn‐seq) (Figure 1a), enables high‐throughput identification of genomic regions involved in the survival of cells when exposed to various conditions (Barquist, Boinett, & Cain, 2013; van Opijnen & Camilli, 2013). A number of organisms have been investigated using this technique, thereby successfully identifying genes important for growth under different stress conditions, essentially by simultaneously investigating the fitness of all single mutants (Gawronski, Wong, Giannoukos, Ward, & Akerley, 2009; Langridge et al, 2009; Lennen & Herrgård, 2014; Santiago et al, 2015). So far, a Tn‐seq method has, however, not been developed for P. putida .…”

Essential Genes Predicted in the Genome of Rubrivivax gelatinosus

“…There were very few essential genes from any of the glycolytic pathways. This result is not unusual; few glycolytic genes are essential in other organisms (13)(14)(15)(16)(17). TnSeq is usually performed on rich media containing combinations of tryptone, peptone, and yeast extract; therefore, the principal carbon and energy sources in these media are amino acids and small peptides.…”

“…Mutants are pooled, and high-throughput sequencing is used to map hundreds of thousands of unique insertions; these data are then used to find genes that are essential under mutagenesis conditions by identifying genes that cannot tolerate disruptive insertions. This technique has been used on a number of model bacterial systems (13)(14)(15)(16)(17). Here, TnSeq was applied to R. gelatinosus, not only to investigate its metabolism but also to reveal other unusual features of the organism.…”

“…Organisms with more genomic content tend to have lower essential gene content by percentage. For example, Salmonella typhi has 356 essential ORFs of its total 4,537 (7.8%) (13), Agrobacterium tumefaciens has 372 of 5,460 (6.8%) (15), and Pseudomonas aeruginosa has 352 of 5,678 (6.2%) (16); conversely, Caulobacter crescentus has 480 of 3,876 (12.4%) (14), Brevundimonas subvibrioides has 447 of 3,393 (13.2%) (15), and Streptococcus pyogenes has 241 of 1,785 (13.5%) (20). Another contributing factor to lower essential gene content is the general lack of metabolic genes.…”

“…TnSeq is a method of identifying essential genes in a high-throughput fashion by mutagenizing a target strain with a transposon, generating upwards of over one million transformants (13). Mutants are pooled, and high-throughput sequencing is used to map hundreds of thousands of unique insertions; these data are then used to find genes that are essential under mutagenesis conditions by identifying genes that cannot tolerate disruptive insertions.…”