Essential Genes Discovery in Microorganisms by Transposon-Directed Sequencing (Tn-Seq): Experimental Approaches, Major Goals, and Future Perspectives

Abstract

Essential genes are crucial for microbial viability, playing key roles in both the primary and secondary metabolism. Since mutations in these genes can threaten organism viability, identifying them is challenging. Conditionally essential genes are required only under specific conditions and are important for functions such as virulence, immunity, stress survival, and antibiotic resistance. Transposon-directed sequencing (Tn-Seq) has emerged as a powerful method for identifying both essential and conditionally essential genes. In this review, we explored Tn-Seq workflows, focusing on eubacterial species and some yeast species. A comparison of 14 eubacteria species revealed 133 conserved essential genes, including those involved in cell division (e.g., ftsA, ftsZ), DNA replication (e.g., dnaA, dnaE), ribosomal function, cell wall synthesis (e.g., murB, murC), and amino acid synthesis (e.g., alaS, argS). Many other essential genes lack clear orthologues across different microorganisms, making them specific to each organism studied. Conditionally essential genes were identified in 18 bacterial species grown under various conditions, but their conservation was low, reflecting dependence on specific environments and microorganisms. Advances in Tn-Seq are expected to reveal more essential genes in the near future, deepening our understanding of microbial biology and enhancing our ability to manipulate microbial growth, as well as both the primary and secondary metabolism.

Keywords: Tn-Seq; essential genes; transposon.

Figure 1

Outline of the two classes of transposable elements. (a) Retrotransposons (Class I). (b) DNA transposons (Class II). Upper panels illustrate the most common retrotransposon and transposon types. Lower panels illustrate the “copy and paste” and “cut and paste” mechanisms.

Figure 2

Tn-Seq procedure based on type IIs restriction enzymes. (a) MmeI digestion. (b) A1 Illumina adapter binding. (c) PCR and A2 Illumina adapter binding (arrows indicate PCR primers). (d) NGS. The MmeI recognition site is located within the inverted transposon repeats. MmeI cuts 20 bp downstream from the two terminal inverted repeats, releasing the transposon with 20 nucleotides of chromosomal DNA at each end. The transposon sequence is shown in blue, inverted repeats in dark blue; Illumina adapter 1 (A1) is in orange; Illumina adapter 2 (A2) is in green.

Figure 3

Tn-Seq procedure based on the circle method. (a) The DNA from the mutant library is randomly fragmented, and Illumina adapter A1 is ligated to both ends. (b) Digestion with a restriction enzyme that cuts within the transposon. (c) Selection of DNA fragments containing the transposon using the Gene Collector technique, which includes DNA ligation and the digestion of single-stranded DNA with an exonuclease [61]. (d) PCR and addition of the A2 Illumina adapter. (e) Next-generation sequencing. The transposon is shown in blue, with its inverted repeats in dark blue; Illumina sequencing adapter 1 (A1) is in orange; adapter 2 (A2) is in green.

Figure 4

Tn-Seq procedure based on PCR with random primers. (a) A first PCR is performed using a transposon-specific primer and various random primers that incorporate the Illumina adapter 2 sequence. (b) A second PCR is carried out with a primer that incorporates Illumina adapter 1 and another primer that hybridises with adapter 2. (c) Next-generation sequencing. Adapter 1 (A1) is shown in orange; adapter 2 (A2) is shown in green; the Tn5 transposon is shown in blue, with the inverted repeats in dark blue.

Figure 5

Tn-Seq procedure based on sonication and Illumina adapter ligation. (a) The DNA from the mutant library is randomly fragmented by sonication. (b) Both Illumina adapters are ligated to the fragments. (c) A PCR is performed with a primer that hybridises with adapter 1 and part of the transposon sequence, and with a primer that hybridises with adapter 2. Arrows indicate PCR primers. (d) Next-generation sequencing. The transposon is shown in blue, with its inverted repeats in dark blue; Illumina adapter 1 (A1) is shown in orange; Illumina adapter 2 (A2) is shown in green.

Figure 6

C-tailing method using linear PCR (a–d) or random fragmentation (e–h). (a) An initial PCR is performed with a single primer specific to the transposon, resulting in single-stranded DNA. (b) Cytosine tail is added. (c) A second PCR is performed using a poly-G primer that incorporates the adapter 1 sequence and a transposon-specific primer that incorporates the adapter 2 sequence. (d) Next-generation sequencing. (e) Random chromosomal DNA fragmentation. (f) Poly-C tailing. (g) PCR using a poly-G primer, a transposon-specific primer. Illumina adapters are incorporated in the primers. (h) Next-generation sequencing. The transposon is shown in blue, with its inverted repeats in dark blue; the Illumina adapter 1 sequence (A1) is in orange; the Illumina adapter 2 sequence (A2) is in green.

Figure 7

Random barcode transposon sequencing. (a) A transposon library with random barcodes is created. (b) A saturated mutant library is created using the transposon library harbouring the barcodes. Each gene in which an insertion occurs is associated with a specific barcode. (c) After sequencing by Tn-Seq, the barcode for each gene is identified. (d,e) This mutant library can be used to perform cultures under different conditions and analyse the dynamics of each gene based on its specific barcode. Conditionally essential genes can be identified using this methodology. The transposon is shown in blue, with the inverted repeats in dark blue; the light pink represents the 20 bp random sequence corresponding to the “barcode”; the dark pink represents the sequences for universal primers 1 (P1) and 2 (P2).

Figure 8

Essential genes identified by Tn-Seq in more than one of the fourteen bacteria shown in Table 1. Grey square indicates that the gene has been identified as essential in at least two bacteria. 1 Rhodobacter sphaeroides [17]; 2 Streptococcus agalactiae [28]; 3 Rhodopseudomonas palustris [31]; 4 Caulobacter crescentus [34]; 5 Azoarcus olearius [33]; 6 Porphyromonas gingivalis [38]; 7 Herbaspirillum seropedicae [30]; 8 E. coli [35]; 9 Mycobacterium tuberculosis [36]; 10 Salmonella typhimurium [11]; 11 Streptococcus pyogenes [5]; 12 Streptococcus pneumoniae [27]; 13 Ralstonia solanacearum [26]; 14 Burkholderia cenocepacia [39]. The bacteria are arranged based on their number of essential genes, with Rhodobacter sphaeroides having the most essential genes in the table and Burkholderia cenocepacia the fewest.

Figure 9

Gene Ontology classification of the essential genes identified by Tn-Seq in the 14 bacterial species shown in Table 1 and Figure 8. (a) Protein class. (b) Molecular function. Essential genes were classified using Escherichia coli as the reference organism (https://geneontology.org/; accessed on 20 September 2024).

Figure 10

Conditionally essential genes. (a,b) Conditionally essential genes identified by Tn-Seq in more than one of the eighteen bacteria listed in Table 2. Only 7 of these bacteria share conditionally essential genes with others. The grey square indicates that the gene has been identified as conditionally essential in at least 2 bacteria. 1 Escherichia coli [50]; 2 Salmonella enterica [52]; 3 Dickeya dadantii [44]; 4 Pseudomonas aeruginosa [51]; 5 Vibrio cholerae [46]; 6 Mycobacterium tuberculosis [49]; and 7 Staphylococcus aureus [54]. The bacteria are arranged based on their number of conditional essential genes, with E. coli having the most essential genes in the table and S. aureus the fewest. (c) Venn diagram illustrating the limited overlap between the essential genes shown in Figure 8 and the conditionally essential genes shown in Figure 10a,b.

Figure 11

Gene Ontology classification of the conditionally essential genes identified by Tn-Seq in the seven bacteria listed in Figure 10. (a) Protein class. (b) Molecular function. Conditionally essential genes were classified using Escherichia coli as the reference organism (https://geneontology.org/; accessed on 20 September 2024).