Acinetobacter ADP1 as a system for in vivo identification of bacterial coproporphyrinogen III oxidases

Current gene annotation methods based on sequence similarity have greatly expanded the ability to search growing databases of genomic information. This system of identification falls short when novel genes are involved or when large protein families show significant homology across an array of protein functions. Such is the case with the bacterial oxygen-independent coproporphyrinogen III oxidase (CPO) HemN. Its association with the radical-SAM protein superfamily has led to incorrect annotation of non-HemN proteins. Radical-SAM family proteins are highly conserved and traditional sequence comparison proves near impossible for accurate identification of HemN proteins. An in vivo system was developed in the naturally transformable bacteria Acinetobacter ADP1. The wild-type CPO was knocked out using basic PCR cloning techniques utilizing this organisms high competency for natural transformation and recombination. The resulting CPO knockout is presented as an in vivo system for identification of bacterial CPOs.