Pasteurella multocida is a Gram-negative coccobacillus that is both a normal commensal and a primary pathogen of a wide range of mammals and birds. P. multocida is the causative agent of several different diseases in different animals that collectively have a large economic impact on global agricultural industries. To better understand P. multocida pathogenic mechanisms, we optimised Transposon-directed insertion site sequencing (TraDIS) for use in P. multocida. TraDIS allows for identification of genomic regions essential for bacterial growth in any condition of interest.
Using the Himar1 transposon we produced a TraDIS library with 81,927 unique insertions in P. multocida strain VP161 (average insertion every 28 bp). We then used this library to screen for essential genes during growth in rich media and growth in chicken serum, and also to select for genes essential for polysaccharide capsule production. Selection in rich medium identified 481 essential genes; 454 (94%) of these had homologs in the database of essential genes. Selection in chicken serum identified 488 essential genes, of which 40 were essential for growth in chicken serum but not rich medium. Selection for non-capsulated mutants via Percoll gradient centrifugation identified genes likely to be required for capsule production. These included previously identified capsule biosynthesis genes and transcriptional regulators (fis and hfq), as well as numerous genes not previously associated with capsule production. Confirmation of genes identified as essential for each condition is ongoing via directed mutagenesis. These analyses will allow for the design of novel therapeutic strategies to combat P. multocida infections.