Genome scanning for conditionally essential genes in Salmonella enterica Serotype Typhimurium

Abstract

As more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5 transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturating Salmonella enterica serotype Typhimurium mutant library recovered from selection under 3 different in vitro growth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes in S. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD, glnL, recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other approximately 70 bacterial species for which EZ:Tn5 mutagenesis has been established.

Fig 1

Schematic diagrams for the Tn-seq method used in this study. (A) A single nucleotide was changed in one mosaic end (ME) of EZ:Tn5 to introduce the BsmFI recognition site (5′-GGGAC(N)₁₀↓-3′/3′-CCCTG(N)₁₄↑-5′). (B) Deep profiling of Tn5-junction sequences by Illumina sequencing.

Fig 2

Identification of the genes conditionally essential for fitness under 3 different selective conditions: dLB, LB-bile, and LB-42°C. Genome-wide view of the fitness index⁻¹ (= total read counts in the input pool/total read counts in the normalized output pool) is shown for each gene identified under the optimal growth condition (LB medium; control) and the 3 different selective conditions. The fitness index⁻¹ was used for the y axis (instead of the fitness index) for better visualization of mutants with a fitness defect, as indicated by the high peaks. The cutoff line of 5 (= 0.2⁻¹) used in this study to determine the fitness defect is shown (dashed lines). The genes selected in this study for further mutant characterization are shown by arrows.

Fig 3

Conditionally essential genes. (A) The genes identified as conditionally essential for fitness under each of the three selective conditions. The numbers of genes that are also essential for optimal fitness in LB medium are shown in parentheses. There were 8 additional genes essential for fitness in LB medium, but dispensable for fitness under all 3 selective conditions. (B) Functional classification of the identified genes.

Fig 4

Competition between the wild type and each of the ΔpyrD, ΔglnL, ΔrecD, and ΔSTM14_5307 mutants. For the competition assay, the wild type and each deletion mutant were mixed in a 1:1 ratio and inoculated into 2 different culture conditions (control versus the relevant test condition indicated). The cultures were diluted into respective selective conditions every 24 h for up to 4 to 8 days. The cell numbers were determined for the wild-type (NA^r) and mutant (Km^r) cells each day using LB plates supplemented with the appropriate antibiotics. (A) ΔpyrD (LB versus dLB); (B) ΔglnL (LB versus dLB); (C) ΔrecD (LB at 37°C versus 42°C); and (D) ΔSTM14_5307 (LB at 37°C versus 42°C).

Fig 5

Comparison of the fitness indices obtained by Tn-seq data (see Table S3 in the supplemental material) and a competition assay (CA) for the ΔpyrD, ΔglnL, ΔrecD, and ΔSTM14_5307 mutants. The comparison was made for 2 different culture conditions (control versus relevant test condition indicated) for each mutant. The results of the competition assay (n = 3) were obtained at day 4 (ΔpyrD, ΔglnL, and ΔrecD) or 7 (ΔSTM14-5307).

Fig 6

In vivo functions of the gene products during animal infection. Among the genes conditionally required for fitness under each selective condition, the numbers of the genes required for in vivo fitness during animal infection (mouse versus chicken) through 2 different infection routes (systemic versus enteric infection) are indicated. The numbers of the genes that produce proteins in vivo are also indicated for the mouse infection model using the 2 different routes of infection. The genes also essential for fitness in LB medium were not included in the numbers of the conditionally essential genes.