Htc1 AND Htc2, TWO Halomonas titanicae CHEMORECEPTORS THAT BIND AROMATIC COMPOUNDS

BALMACEDA, R.S.; JUNG, K; STUDDERT, C.A.; GASPEROTTI, A. F.; HERRERA SEITZ, M.K.

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades Científicas 2017; 2017

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Halomonas titanicae KHS3 is a strain isolated from Mar del Plata harbor that is able to grow on aromatic hydrocarbons and displays chemotactic behavior toward them.Genomic sequencing allowed the identification of 25 chemoreceptor genes. Most of the genes code for proteins with a predicted periplasmic ligand-binding domain and a highly conserved cytoplasmic signaling domain. To identify those chemoreceptors involved in sensing of aromatic compounds, we first looked at their genomic context. We found one chemoreceptor, Htc1, next to genes related to degradation of aromatic compounds. The ligand-binding domain of this receptor was expressed in E. coli as a His-tagged protein. The purified protein was subjected to thermal shift assays. In this assay the Tm (melting temperature) of the protein is expected to increase in the presence of stabilizing ligands. Salicylate, 4-hydroxybenzoate and benzoate increased the Tm, among more than 300 compounds present in the screening plates. The binding was confirmed by thermophoresis assays. All three compounds bind to the periplasmic domain of Htc1 with Kds between 80 and 600 uM. Besides, the full-length receptor conferred the ability to control the flagellar movement in response to salicylate when expressed in E coli. An Halomonas titanicae Htc1 mutant, however, showed only subtle alterations in chemotaxis, suggesting that there is functional redundancy between Ht chemoreceptors.Among other ligand binding domains from chemoreceptor genes that were subjected to thermal shift assays we found another one (Htc2) whose Tm was also increased with salicylate, as well as with malate, malonate and succinate. The binding properties were characterized using isothermal calorimetry (ITC). The C3-C4 dicarboxylic acids bound with high affinities (Kds between 30 and 600 uM), whereas salicylate binds poorly (Kd about 2.5 mM). Full-length cloning and expression for functional characterization in E.coli cells and mutant construction are under way.