The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of 24 the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our 25 previous comparative genomic and functional transcriptomic studies. We used a genome-wide 26 transposon library with insertions every 10 nucleotides to identify genes required for survival and growth 27 in vitro and during infection of murine macrophages. The analysis revealed genomic regions important 28 for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal 29 growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal 30 medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 31 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic 32 regions required for growth in at least one in vitro growth condition. By infecting murine macrophages 33 with the transposon library, we identified 68 genes that were required for intra-macrophage replication 34 but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, 35 consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and 36 suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine 37 macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-38 encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other 39 bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our 40 enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge 41 tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA 42 synthetases in plasmids from a range of gram-negative and gram-positive bacteria suggests that 43 plasmid-encoded essential genes are more common than had been appreciated.