The bare necessities: Uncovering essential and condition‐critical genes with transposon sequencing

Abstract

Querying gene function in bacteria has been greatly accelerated by the advent of transposon sequencing (Tn-seq) technologies (related Tn-seq strategies are known as TraDIS, INSeq, RB-TnSeq, and HITS). Pooled populations of transposon mutants are cultured in an environment and next-generation sequencing tools are used to determine areas of the genome that are important for bacterial fitness. In this review we provide an overview of Tn-seq methodologies and discuss how Tn-seq has been applied, or could be applied, to the study of oral microbiology. These applications include studying the essential genome as a means to rationally design therapeutic agents. Tn-seq has also contributed to our understanding of well-studied biological processes in oral bacteria. Other important applications include in vivo pathogenesis studies and use of Tn-seq to probe the molecular basis of microbial interactions. We also highlight recent advancements in techniques that act in synergy with Tn-seq such as clustered regularly interspaced short palindromic repeats (CRISPR) interference and microfluidic chip platforms. K E Y W O R D Sessential genes, functional genomics, next-generation sequencing, oral microbiology, transposon sequencing

A bacterial pan-genome makes gene essentiality strain-dependent and evolvable

“…These genes may thus tolerate more mutations than a core genome gene involved in central processes, such as DNA replication; 3) Functional enrichment analysis shows that universal essential genes are enriched in 5 COG categories (Fig. 2F), all of which support central cellular processes and have been found as enriched in essential gene sets from different species 4, 44, 45 , while strain-dependent and accessory essential genes are enriched for only two of these categories (Fig. 2F); 4) Since gene essentiality for some genes depends on the strain background, we reasoned that they would likely still trigger a growth defect in the strains where they are not essential.…”

“…Essential genes may have characteristics that distinguish them from non-essential genes. For instance, an essential gene’s expression is often higher, and much less affected by environmental changes than the average non-essential gene 1, 2, 4, 43 . We assessed whether the identified essentialome genes have different characteristics compared to non-essential genes: 1) RNA-Seq was performed on the 36-strain PG collection grown in rich medium, which shows that the universal, strain-dependent and accessory essential genes tend to be more highly expressed compared to non-essential genes (Fig.…”

“…In contrast, essential genes are critical for maintaining functionality under any circumstance. This is reflected in their high level of conservation within and often across species, limited genetic diversity, and high and stable expression levels 2–4 . Consequently, essential genes are considered rigid, largely immutable key components to an organism’s survival, which makes them attractive as antimicrobial targets 5, 6 .…”

Current Knowledge and Future Directions in Developing Strategies to Combat Pseudomonas aeruginosa Infection

“…In this strategy, pooled populations of transposon mutants covering the entire genome are cultured in a selected environment. Sequencing tools are then used to pinpoint genomic regions which are important or essential for bacterial fitness [20]. When applied to Pa, this technique has facilitated the rapid identification of genes which are important for fitness in infection-relevant environments, single nutrient sources, energy limitation, antibiotic resistance, competing microorganisms, and motility [21][22][23][24][25][26][27][28][29].…”

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods

“…TnSeq couples genome-wide transposon mutagenesis with high-throughput deep sequencing of the transposongenomic junctions (referred to herein as 'transposon insertions' or 'insertions') to identify genes which compromise bacterial fitness under a given experimental growth condition [5]. Several groups have successfully applied this tool to define putative essential genes of different species [5][6][7][8][9]. For bacterial pathogens, TnSeq has been used to characterize genes required for establishing successful infections [10,11].…”