P71 Using transposon mutagenesis and deep sequencing to identify conditionally essential genes in Rhodobacter sphaeroides
Sunday, August 2, 2015
Brian Burger, Saheed Imam and Tim Donohue, Bacteriology, University of Wisconsin-Madison, Madison, WI
Optimizing microbial growth or end-product synthesis in laboratory or industrial settings requires the coordinated activity of a suite of genes. Next-generation strain engineering can benefit from genome-scale information on the genes that are essential under a given set of growth conditions. This poster describes new, genome-scale insight on conditionally essential genes in Rhodobacter sphaeroidesusing the Tn-seq approach.

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.