Home-made evolution helps to understand the signaling abilities of natural chemoreceptors?

KARINA HERRERA SEITZ; DIEGO A. MASSAZZA,; FLORENCIA GASPEROTTI; CLAUDIA A. STUDDERT

Mar del plata

Congreso; Sociedad Argentina de Microbiología General; 2012

Bacterial chemoreceptors typically possess an extracellular sensing domain and a cytoplasmic signaling domain that transmits the signal to the flagellar motor. The cytoplasmic domain of chemoreceptors is strikingly conserved in Bacteria and Archaea. It consists of a long alpha helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Most of the thousands of chemoreceptors identified in nature can be classified into one of seven receptor classes, defined by the length of their cytoplasmic hairpin. Receptors belonging to different classes differ by the presence of symmetric insertions or deletions of seven-residue stretches (heptads) in both arms of the hairpin. Classes are named according to the total number of heptads that are present in the domain. In order to understand the structural features that are required for a proper transmission of the signal (ligand binding) from the periplasmic domain to the extreme of the cytoplasmic hairpin where the histidine kinase binds, we focused in the comparison between E. coli receptors (36-heptad class) and receptors of the 40 heptad-class. On the one hand, we characterized the signaling abilities of a natural 40H-class receptor from Shewanella oneidensis expressed in E. coli. It was able to control the histidine kinase and respond to nitrate as an attractant, indicating that it assembled functional signaling complexes with E. coli proteins. On the other hand, we introduced symmetric 14-residue insertions, compatible with the coiled-coil organization, into Tsr,the serine chemoreceptor of E. coli, in order to convert it into a receptor of the 40-heptad class. The insertions were introduced at two different locations of the hairpin, and their functional effects were analyzedThe ability to activate the CheA kinase, to modulate its activity in response to serine and the ability to mediate chemotaxis responses in soft agar plates were evaluated in cells expressing the Tsr-derived constructs. Both Tsr derivatives were able to activate the CheA kinase. However, no responses towards serine were detected. Random mutagenesis applied into one of the constructs allowed the isolation of functional derivatives, indicating that subtle changes restored signaling abilities to the enlarged hairpin. A detailed characterization of the 40-heptad class receptors in E. coli is presented, both from a functional point of view and concerning their in vivo organization in the cell.