A genome-wide collection of barcoded single-gene deletion mutants in Salmonella enterica serovar Typhimurium

Abstract

Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences. A motility assay of a pool of the entire library, followed by a single-tube processing of the bacterial pellet, PCR, and sequencing, was used to verify the performance of the barcoded TetR collection. The new resource is useful for experiments with defined subsets of barcoded mutant strains where biological bottlenecks preclude high numbers of founder bacteria, such as in animal infections. The TetR version of the library will also facilitate the construction of triple mutants by transduction. The resource of 6197 mutants covering 3490 genes is deposited at Biological and Emerging Infections Resources (beiresources.org).

Fig 1. Lambda red recombination of barcodes into existing single-gene deletion mutants.

A PCR product containing homology to both ends of the existing mutant cassette of non-barcoded mutants (green and pink lines) is recombined to produce tetracycline-resistant mutant clones with 21-base barcodes (BC1 and BC2), one of which is flanked by Illumina Read sequences. See S1 Fig for the complete sequence of the replacement cassette, and Materials and Methods for details.

Fig 2. Workflow of the iterative conversion of the non-barcoded single-gene deletion (SGD) resource to barcoded Tet^R SGD collection.

Conversion of the Kan^R resource is depicted. See text for details.

Fig 3. Determination of integration sites associated with specific barcodes in SGD libraries by Illumina sequencing after Klenow / PCR amplification.

See Materials and Methods for details, including primer sequences.

Fig 4. Differences in mutant representation in the ring vs the center of motility agar plates after inoculation with a pool of 6,207 barcoded single-gene deletion mutants.

See Materials & Methods for details. Each dot depicts a gene, usually represented by two mutants which were combined in the statistical analysis. Underlying data in single-mutant resolution are shown in S5 Table. Blue = p_adj < 0.05; grey = p_adj > 0.05. Some notable statistically significant genes are annotated. Data are from four separate experiments.