The fitness landscape of the AfricanSalmonellaTyphimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid

AbstractWe have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the AfricanSalmonella entericaserovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growthin vitroand during infection of murine macrophages. The analysis revealed genomic regions important for fitness under twoin vitrogrowth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of otherS.Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least onein vitrogrowth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitnessin vitro. None of these genes were unique toS.Typhimurium D23580, consistent with a high conservation of gene function betweenS.Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1had a lower ability to charge tRNA than the chromosomally-encoded CysRSchrenzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of gram-negative and gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.