Title: Deletion of the Rnf Operon in Vibrio Cholerae

ABSTRACT: The NADH: ferredoxin oxidoreductase (Rnf) from Vibrio cholerae is encoded by the rnf operon which comprises 6 genes (rnf A,B,C,D,G,E). The gene products make up the Rnf complex, a membrane protein made up of 6 subunits (Rnf A, B, C, D, G, and E). Rnf has been studied in Rhodobacter capsulatus, where this enzyme is essential for nitrogen fixation. However, now that many bacterial genomes have been completely sequenced, we have found that the rnf operon is widely distributed among many kinds of bacteria, including marine and soil bacteria, strict anaerobes and some pathogens such as Vibrio cholerae. Furthermore, Rnf shares significantly homology with the Na+-pumping NADH: quinone oxidoreductase, which is the first electron carrier in the respiratory chain of many marine and pathogenic bacteria. As a first step towards studying Rnf we need to delete the genomic copy of the rnf operon and create a recombinant expression system. Our initial attempts to delete the rnf operon suggested that the rnf genes are essential for survival of Vibrio cholerae. For this reason, we are now using a rescue-plasmid strategy for the deletion. We have built a deletion construct using a suicide vector whereby a double crossing over event will result in excision of the genomic copy of rnf. We have also built a rescue plasmid, which will allow Rnf to be expressed in trans under control of the arabinose promoter, while the genomic copy is being deleted.