Characterization of the twin arginine transport pathway in the plant symbiont Sinorhizobium meliloti

Sinorhizobium meliloti is an alpha-proteobacterium that is capable of living both saprophytically as well as in association with alfalfa, sweet clover or Trigonella. In this work we have initiated a characterization of the role of the twin-arginine transport system in S. meliloti.In this work we show that Tat appears to be essential to the survival of S. meliloti. Since we were unable to create a viable tat strain we selected a subset of predicted Tat substrates and constructed 30 deletion mutants. Screening of these mutants on alfalfa and sweet clover showed that some of these had a differential effect of their plant hosts.A more in depth investigation of a previously observed phenotype suggested FdoG contains a Twin-arginine leader signal sequence. This observation led to the characterization of formate dependant autotrophic growth in S. meliloti using both physiological and molecular techniques.Finally, we addressed the hypothesis that leader binding proteins exist in S. meliloti. Using a chimeric FdoGleader/malE protein fusion for pulldown assays with protein extract isolated from autotrophically grown S. meliloti, several putative leader binding proteins were identified.The twin-arginine transport (Tat) pathway is a protein secretion system that is noted for its ability to transport proteins that have been folded within the cytoplasm. Since many of the proteins tend to be periplasmic redox enzymes, our initial goal was to create a tat mutation and quantitate its role in bacterial/plant interaction.Taken together, these results allow us to propose that the twin-arginine transport pathway in S. meliloti plays a central role in the cell. This work provides the groundwork for subsequent research of the Tat pathway in S. meliloti.