Analysis of components of the chemotaxis signaling complex through FRET-based in vivo assays and photoactivated localization microscopy (PALM technology)

ANDREA PEDETTA; JOHN S. PARKINSON; CLAUDIA STUDDERT

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

Sociedad Argentina de Microbiología General

Bacterial chemotaxis allows microorganisms to sense their immediate chemical environment and modulate their movement accordingly. The main components of this signal transduction system are chemoreceptors, which sense the chemical stimuli and control the activity of the histidine kinase CheA, which in turns transmits the signal to the flagellar motors. The small protein CheW, structurally homologous to one of the domains of CheA, couples the receptors with the kinase, playing an essential role in CheA control. The aim of our work is to understand the precise role of CheW, which seems to be more complex than that of a simple bridge between the two proteins. We focused our study in the conserved residues R62 in CheW and R555 in CheA. These arginine residues lie in equivalent positions in CheW and the homologous domain in CheA, and it has been shown that replacements in any of them result in severe impairment of the chemotactic behavior. However, the mutant proteins do not display evident defects in function when studied in vitro, using chemoreceptor-containing membranes, or in kinase control in vivo, suggesting that the alteration might affect in a general way the organization of the polar chemoreceptor cluster. In this study, we use two different assays intended to assess the effect of CheW or CheA mutant proteins with replacements in R62 or R555 on the assembly of polar receptor clusters and on their signaling ability. We performed a FRET-based assay to measure the changes in the kinase activity when cells are exposed to different concentration of attractants, which allow us to determine whether the sensitivity and cooperativity of the system are affected. Whereas in CheW-R62 mutants the sensitivity was not affected, the cooperativity was reduced. In CheA-R555 mutants both parameters showed to be more affected. Photoactivated localization microscopy (PALM) is a powerful technology that permits a detailed analysis of the number of chemoreceptors that form part of a single cluster, as well as the number and localization of chemoreceptor clusters in a single cell. Using this technology we did not find significant differences between the clusters containing wild-type or mutant versions of CheW. The effect of the more disturbing mutations in CheA on cluster organization remains to be determined. Taken together, these results suggest that small changes in the conformation of the ternary complexes may lead to reduced sensitivity and/or cooperativity of the system. These differences might cause a failure in the amplification of the signal, which finally would affect the chemotactic response much more than expected.