Projects:

    The ssrA gene encodes tmRNA, a transfer-messenger RNA that acts on ribosomes stalled during translation, tagging the nascent polypeptide for proteolysis. Deletion of ssrA in Caulobacter crescentus results in a delay of DNA replication initiation. Additionally, deletion of ssrA or smpB, which encodes a protein that protects tmRNA from degradation, causes a disruption of plasmid maintenance. This plasmid maintenance phenotype is of interest since tmRNA is present in all sequenced bacterial genomes, and plasmids are known to harbor genes important in development and virulence. Several broad host range plasmids including members of the pBBR and RSF1010 families are able to replicate in wild type C. crescentus. However, strains lacking tmRNA activity due to loss of ssrA or smpB can no longer be stably transformed with any of these plasmids, unless the plasmid carries a gene that complements the chromosomal mutation. Plasmids containing the C. crescentus origin of replication are maintained in cells lacking tmRNA activity, indicating that there are no gross defects in plasmid physiology. Two selection strategies were used to find suppressors of the plasmid maintenance phenotype. A pBBR1-based plasmid was mutagenized, and mutations that bypass the need for tmRNA activity were selected. A deletion that results in a truncation of the C-terminal end of Rep, the origin-binding protein of pBBR1, allows the plasmid to be maintained in the presence or absence of tmRNA activity. Wild-type Rep was also identified as a substrate for tmRNA. These data led to the hypothesis that Rep must be processed by tmRNA for plasmid replication to proceed normally. tmRNA may also interact with proteins encoded by chromosomal genes to control plasmid maintenance. A selection for chromosome-based mutations that allow maintenance of pBBR1-based plasmids has revealed a suppressor of the plasmid maintenance phenotype. This second-site suppressor grows at the same rate as the ssrA deletion strain, indicating that it does not suppress the chromosome replication phenotype. These data imply that the DNA replication initiation delay and the plasmid maintenance phenotypes are caused by distinct regulatory effects rather than a global defect, and suggest that tmRNA may have specific roles in DNA replication initiation and plasmid maintenance that are separate from protein quality control.