INTEMA   05428
INSTITUTO DE INVESTIGACIONES EN CIENCIA Y TECNOLOGIA DE MATERIALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
AN ENGINEERED SERINE CHEMORECEPTOR WITH SYMMETRIC INSERTIONS MIMICS NATURAL TRANSDUCERS OF THE 40H CLASS
Autor/es:
KARINA HERRERA SEITZ; DIEGO A. MASSAZZA,; CLAUDIA A. STUDDERT
Lugar:
Tucson, Arizona
Reunión:
Congreso; BLAST XII (Bacterial Locomotion and Signal Transduction ); 2013
Resumen:
Bacterial chemoreceptors usually detect extracellular signals through a periplasmic sensing domain and transmit them to a highly conserved intracellular domain. The signal then reaches the flagellar motors to control swimming behavior. The cytoplasmic signaling domain consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. The huge variety of chemoreceptors identified from genomic analysis in Bacteria and Archaea can be classified into a small number of classes according to the length of their cytoplasmic signaling domain. Differences in length are due to the presence of pairs of insertions or deletions of seven-residue stretches (heptads), located symmetrically with respect to the hairpin turn. The size and location of the indels highlight the importance of the coiled-coil structure and suggest the existence of specific interactions between the two arms of the hairpin, that are needed to preserve proper signal transmission. To understand the structural requirements of signal transmission that led to this peculiar evolution pattern, in our lab we are engaged in the construction and characterization of derivatives of the serine chemoreceptor of E. coli, Tsr (36H-class because it possesses 36 heptads in its cytoplasmic domain), to mimic natural transducers of different length classes. In this work, we built a 40H-class derivative of Tsr through the introduction of symmetric 14-residue insertions, whose sequence and position were chosen based on the alignment of Tsr with PctApp, a 40H-class receptor from Pseudomonas putida. The 40H-class derivative, TsrH18, was able to activate the CheA kinase at a high level. However, it was insensitive to serine and unable to mediate serine taxis in soft agar plates. Random mutagenesis applied to the construct allowed the isolation of two functional derivatives (TsrH18*) that carry single point mutations, indicating that subtle changes restored signaling abilities to the enlarged hairpin. TsrH18* derivatives were able to control CheA activity in response to serine. They also localized to the poles of the cell, as assessed with the fluorescent reporters YFP-CheZ or YFP-CheR. In contrast, the original TsrH18 construct failed to mediate YFP-CheZ localization, suggesting its inability to form properly assembled ternary complexes. Unlike the derivative belonging to the 34H class that he had characterized in previous work, this 40H-class derivative does not interfere with the chemotactic function of native Tar (36H class), and it does not form mixed trimers of dimers with Tar, probably due to the larger length difference. The availability of functional derivatives that mimic receptors of different classes represent useful tools to study interactions between different receptors in the same cell and the requirements for co-operation within the same chemoreceptor cluster.