Transposon Insertion Sequencing to eastablish the fitness landscape of the African Salmonella Typhimurium ST313 strain D23580

To complement our previous comparative genomic and functional transcriptomic studies, we used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under in vitro growth conditions involving LB nutrient medium and an acidic minimal medium that induced expression of the SPI-2 type-three secretion system. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing media. These findings correlated well with the essentiality analyses that have been performed in S. Typhimurium ST19 and S. Typhi strains. The genome-wide analysis identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that strain D23580 does not possess novel virulence factors involved in the infection of murine macrophages. We discovered that a high number of pBT1 plasmid-encoded genes (36) rarely had transposon insertions, compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. Our enzymological analysis revealed that CysRSpBT1, the key essential protein encoded by pBT1, had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of gram-negative bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.