| Proteolysis of Phd is required for the addictive response. ClpXP recognizes and degrades the Phd protein of the P1 plasmid addiction operon, however its mechanism of degradation is currently unknown. The RssB and SspB proteins aid ClpXP in degrading other substrates, so these might also play a role in the proteolysis of Phd. A temperature sensitive plasmid was introduced with and without the P1 plasmid addiction operon to strains containing knockout mutants of the rssB and sspB genes. An addiction assay was performed using these constructs by incubating cells at restrictive and permissive temperatures for plasmid replication. Mutations in rssB and/or sspB did not relieve addiction indicating that neither RssB nor SspB are required for proteolysis of Phd by ClpXP. beta-galactosidase assays on strains with and without ClpXP indicated that degradation was only observed when there were low levels of Phd present suggesting that Phd is diluted before it is degraded. |
