“A putative aminoacid chemoreceptor of P.putida: cloning, expression and functional analysis in E.coli”

DÉBORA SOTO; KARINA HERRERA SEITZ; CLAUDIA STUDDERT

Carlos Paz, Córdoba

Congreso; VI jornadas de SAMIGE (Sociedad Argentina de Microbiología); 2009

SAMIGE (Sociedad Argentina de Microbiologia General)

The signal transduction pathway involved in chemotaxis is present with little variations along bacteria and archaea. Chemoreceptor genes, coding for membrane proteins that sense environmental stimuli and transduce the signal to the flagellar motors, are easily recognized in genomic studies due to a highly conserved signaling domain. This domain is implicated in the interaction with the histidin kinase whose activity is modulated in response to stimuli. En E.coli chemoreceptors of same or different specificities are organized in trimers of dimers. Dimer to dimer interactions involves a region of the signaling domain. It is not well established, however, wheter this organization is a conserved feature that might be central to chemoreceptor function. To address this function, we cloned chemoreceptors genes from different organisms and expressed them in E.coli to carry out structural and functional studies. Here, we report cloning and expression of a putative aminoacid receptor from P.putida. As a first approach to address if chemoreceptors of microorganisms non-E.coli are also arranged in trimers of dimers, cloning and expression of a chemoreceptor of P.putida KT2440 (pctApp) was carried out. The cloned gene was nicely expressed and recognized by antibodies raised against the Tsr conserved cytoplasmatic domain from E.coli. Several experimental approaches, including Western blot analysis, crosslinking assays, rotational analysis and swimming plates were used to check the function of this gene in E.coli cells. The results showed that pctApp was not able to complement Tsr in soft agar plates, however, pctApp could activate the kinase and respond to stimuli in tethering assays. In crosslinking assays pctApp seems to fail to form stable mixed trimers.