Genetic transformation of ammonia-oxidizing bacteria

The study of traits of ammonia-oxidizing bacteria (AOB) by genetic transformation is an approach that is facilitated by the availability of AOB genome sequences. To transform an AOB, a vector construct is introduced into the cells by electroporation or conjugation to effect the inactivation, complementation, or expression of a selected gene. For inactivation studies, the vector construct should contain the gene of interest with an antibiotic resistance cassette and recombine into the cell's chromosome. For gene expression studies, a wide-host range vector with a transcriptional gene fusion can be used to test for gene roles. For gene complementation studies, a wide-host range vector expressing the inactivated gene can be used to recover the lost function in an AOB mutant strain. This chapter is a compilation of the methods that have been used to transform the AOB Nitrosomonas europaea and Nitrosospira multiformis and of the considerations and caveats to successfully produce, maintain, and store AOB transformants. The protocols may be applied to other AOB.