Tn-seq combines traditional transposon mutagenesis with the power of deep sequencing, allowing identification of transposon insertion sites in a mutagenized population en masse. Monitoring changes in transposon mutant abundance at the gene level before and after growth in a specified condition allows one to assign value to the importance of individual genes. Thus, with a saturated transposon mutant library and sufficient sequencing depth, both essential genes and fitness contributions of non-essential genes can be determined on a genome-wide scale.
We deployed Tn-seq in Rhodobacter sphaeroides, a facultative purple non-sulfur bacterium that is used industrially to produce hydrophobic compounds for use as fuels or chemicals. We used Illumina sequencing to identify insertion sites in a ~200,000-member transposon mutant library under a variety of laboratory growth conditions. The resultant data allowed us to define, for the first time, the essential genome of this bacterium as we vary either media composition or growth conditions, expanding our knowledge of genes required for growth. Moreover, the identification of genes contributing differential fitness effects has contributed a new understanding of how cells respond to stress generated during solar energy conservation, respiration, metabolism of plant hydrolysates, or biofuel production.