High-Throughput Mutant Screening via Transposon Sequencing

INTRODUCTION

In the 1970s, researchers began using transposons as tools “to manipulate the genes of bacteria, bacteriophage (phage), and plasmids in ways which are otherwise difficult or impossible” (Kleckner et al. 1977). Transposons are genetic elements that can move within or between genomes by one of two mechanisms: replicative (conservative) transposition (e.g., Tn3), in which the transposon is duplicated (Shapiro 1979), or nonreplicative (cut-and-paste) transposition (Bender and Kleckner 1986) (e.g., Tn10). Both mechanisms are mediated by a transposase enzyme that recognizes inverted repeat sequences at the ends of the transposon, and then locates a target sequence for insertion of the transposon (Craig 1997). For some transposons, insertion occurs in random sites, whereas others are pseudorandom or nonrandom. Random insertion is necessary for performing mutant screens and selections. Transposons naturally encode their own transposase. By contrast, mini-transposons (e.g., the mTn10 derivative used in Protocol: High-Throughput Mutant Screening in Vibrio cholerae via Transposon Sequencing [Bourgeois and Camilli 2023]) are engineered to have inverted repeats flanking an antibiotic-resistance gene but lack the transposase gene. The transposase must be provided in trans, typically on a plasmid, to mediate transposition. The advantage of mini-transposons is that insertion mutants are completely stable once the transposase gene is no longer expressed or present in the cell.

Traditionally, researchers would follow up a transposon mutant screen or selection by analyzing one or, at most, a handful of mutants with interesting phenotypes. However, in 2009, three groups began combining transposon mutagenesis with massively parallel sequencing (MPS) of the transposon junctions to allow for high-throughput, genome-wide screens and selections (for review, see van Opijnen and Camilli 2013). These methods, often referred to as transposon sequencing (Tn-seq), monitor changes in the frequency of individual mutants within a saturated transposon insertion library, allowing the fitness contribution of virtually every gene in a genome to be assessed in a single experiment.

As a bonus, Tn-seq also identifies the essential gene set of a microorganism. Essential genes are identified by virtue of lack of transposon insertions in an otherwise saturated library. Confirmation of gene essentiality requires additional experiments, such as deleting the chromosomal copy of the gene while providing the gene in trans on a plasmid under the control of an inducible promoter and then showing loss of viability upon removal of inducer. Ideally, the transposon mutant library is constructed during growth of the microorganism in a rich medium, in order to limit the number of essential genes. In this way, conditionally essential genes can be identified in more challenging growth conditions.

The user can apply the Tn-seq approach to any microorganism for which transposition or other forms of insertional mutagenesis are established, such as suicide plasmid integration or CRISPR–Cas-directed gene replacement or insertional disruption. Once an insertional mutant library is constructed and cryopreserved, the user can subject an aliquot of the library to any selection condition of interest, including infecting animals in the case of pathogens. Either conditions of growth, or stress conditions, or a combination of the two, can be utilized.

One important consideration for any Tn-seq experiment is avoiding bottlenecks during the procedures, particularly during the selection condition. The reason for this is that if many transposon insertion mutants disappear stochastically from the population because of the bottleneck, the user cannot know whether a gene is conditionally essential or not. For example, if the library is used to infect tissue culture cells or an animal model of infection, but only a fraction of the inoculated cells has the opportunity to colonize, then most of the population will be eliminated stochastically. Another example is exposing the library to such a harsh environment that most of the population dies immediately without having the chance to adapt. Bottlenecks of >20% of the starting transposon insertion mutants (library complexity) typically result in a failed experiment. Methods to alleviate the impact of bottlenecks include reducing the library complexity going into the selection condition, changing the selection condition to reduce the bottleneck effect, or preadapting the input population to reduce stochastic loss of cells in the selection.

In our associated protocol, we introduce procedures for using one of the Tn-seq methods to perform a genome-wide selection for Vibrio cholerae genes involved in growth on chitin (see Protocol: High-Throughput Mutant Screening in Vibrio cholerae via Transposon Sequencing [Bourgeois and Camilli 2023]). V. cholerae is a Gram-negative bacterium that lives in temperate estuarine environments around the world but is also a human pathogen and causative agent of cholera. Putatively essential genes for general growth in a rich medium will be identified first. Next, the fitness contribution of nonessential genes will be measured during growth in an estuarine-like condition in which chitin serves as the sole carbon and nitrogen source.

The first procedure in the accompanying protocol is for constructing a saturated mini-transposon library using in vivo transposition. Next is a procedure for passaging the library on a growth condition of interest. This is followed by procedures for isolation of genomic DNA and then capture of the transposon–genome junctions using the homopolymer tail-mediated polymerase chain reaction (PCR) (van Opijnen et al. 2015) method. After this, MPS is conducted on the Illumina NextSeq instrument. Last, the accompanying protocol has a procedure for analyzing the MPS data using a custom script written in the Python programming language to identify genes that contribute measurably to growth under the selection condition.