Fig. 3.

In vivo cross-linking properties of prototype interface 2 mutants. (A) Cells of strain UU2806 [∆(cheA-cheZ) ∆(tar, tap, tsr, trg, aer)] carried two compatible plasmids: pRR53 to express the Tsr receptor at different levels and a derivative of pPM25 expressing an HA-tagged CheA-A546C/CheW-E27C cross-linking reporter pair in combination with an interface 2 lesion (CheA-L545S, V551A, Y558G, and CheW-X3). Cells were grown and treated as detailed in Materials and Methods, and lysate proteins were separated by SDS/PAGE and probed with anti-HA antibody to detect cross-linked CheA∼CheW products. (B) The band profiles shown in A were quantified by densitometry, and the fraction of CheA cross-linked to CheW in each experiment was normalized to the cross-linking yield at each Tsr expression level for reporter proteins bearing no interface 2 lesions. Histogram bars show the mean and SE for between three and five independent experiments.