IDENTIFICATION AND CHARACTERIZATION OF Tsr MUTANTS THAT ARE DEFECTIVE IN

DIEGO A. MASSAZZA; JOHN S. PARKINSON; CLAUDIA A. STUDDERT

. Laughlin, Nevada, USA

Congreso; BLAST IX (Bacterial Locomotion And Signal Trasduction); 2007

E. coli chemoreceptors appear to be organized in trimer-of-dimer arrangements that may be important to their signaling activities. We have developed an in vivo crosslinking competition assay to identify mutant chemoreceptors with defects in trimer formation. In the competition assay, serine receptors (Tsr) are expressed at low or high level in cells expressing chromosomal levels of an aspartate receptor bearing a cysteine reporter near the trimer axis (Tar?C). The cells are then treated with the tri-functional maleimide-targeted crosslinker TMEA and the extent of Tar?C crosslinking is measured in the presence of low and high levels of the Tsr ?competitor.? Trimer-proficient Tsr molecules cause a dramatic decrease in Tar?C crosslinking products when expressed at high levels, whereas trimer-defective Tsr molecules do not interfere with the formation of Tar?C crosslinking products. Using this competition assay, we identified several trimer-defective Tsr mutants based on their inability to dilute crosslinking yields of the Tar?C reporter molecules. Quantitative analysis of the competition assay allowed us to identify Tsr variants that are completely unable to interact with Tar-C dimers in mixed trimers, as well as ones that seem to be incorporated into mixed trimers as single dimers but which show difficulty in contributing two dimers to the same trimer. To further characterize the trimer formation ability of the Tsr mutants, we introduced a cysteine reporter into each of them and analyzed their direct TMEA-crosslinking behavior when expressed as the only receptor in the cell or together with Tar?C. Direct crosslinking assays of the Tsr mutants were generally consistent with the behavior predicted from the competition assay: some Tsr mutants showed a clear decrease in their ability to get crosslinked and some showed the expected bias in forming mixed crosslinking product/s with Tar-C. However, the competition assay seemed to be more sensitive to trimer formation defects, presumably due to the fact that direct crosslinking can potentially trap transient, unstable species.