ERP133374 PRJEB48933 6345830a-d2e4-4e29-9e04-4c74b6917005 The genetic determinants for survival of Mycobacterium bovis in cattle Transposon libraries have been effectively used in the context of human adapted Mycobacterium tuberculosis to identify the genetic basis of survival in murine models. However, there have been limited studies examining other members of the MTBC. In this study we use a high-density transposon library to determine the genes required for survival of Mycobacterium bovis in cattle. We show that genes involved in PDIM biosynthesis, the ESX-1 secretion system, cholesterol catabolism and mycobactin biosynthesis are required in vivo in cattle, corroborating current knowledge. However, we also identify several novel genes that are required in vivo, such as glpD2, a gene involved in glycerol-phosphate metabolism. The work presented here represents the first genome-wide study of gene essentiality in a member of the MTBC within a natural host and offers novel insights into genetic basis of survival during infection. EradbTB Transposon libraries have been effectively used in the context of human adapted Mycobacterium tuberculosis to identify the genetic basis of survival in murine models. However, there have been limited studies examining other members of the MTBC. In this study we use a high-density transposon library to determine the genes required for survival of Mycobacterium bovis in cattle. We show that genes involved in PDIM biosynthesis, the ESX-1 secretion system, cholesterol catabolism and mycobactin biosynthesis are required in vivo in cattle, corroborating current knowledge. However, we also identify several novel genes that are required in vivo, such as glpD2, a gene involved in glycerol-phosphate metabolism. The work presented here represents the first genome-wide study of gene essentiality in a member of the MTBC within a natural host and offers novel insights into genetic basis of survival during infection. ENA-FIRST-PUBLIC 2022-01-31 ENA-LAST-UPDATE 2022-01-31