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

PEDETTA, A; PARKINSON, J. S.; STUDDERT, C.A.

Mar del Plata

Congreso; Octavo Congreso Argentino de Microbiología General (Sociedad Argentina de Microbiología General, SAMIGE); 2012

SAMIGE

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 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 that 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 clusteris central to its role. A particular R62 mutant protein (CheW-R62H) showed that, despite being defective for chemotaxis in soft agar plates, its binding to receptors and to CheA and its ability to activate the kinase in vitro are similar to wild-type CheW. In a first attempt to determine whether the defect was only visible in in vivo assays, we measured its ability to activate CheA in vivo, as well as the ability to form ternary complexes in the pole of the cells. However, we were not able to find notorious differences with wild-type CheW protein (SAIB 2010). Further characterization of two CheW-R62 mutants (R62H and R62C) was done using FRET and PALM technologies. FRET assays allow us to determine whether the sensitivity and cooperativity of the system is affected. CheW-R62 mutants showed that, whereas the sensitivity may be not be affected, the cooperativity is reduced. PALM technology was used in order to analyze quantitatively the ability of the mutants to form ternary complexes in the poles of the cell. Preliminary results did not show differences between mutants and the wild-type protein, but further analyses are required. The results obtained to the present show that CheW-R62 mutant proteins, in spite of not showing notorious differences in each step of the signaling mechanism when compared to wild-type proteins, may be affected in the cooperativity of the system. This might indicate that small changes in the conformation of the ternary complexes can affect the chemotactic response more than expected.