The Wsp-like gene cluster of Halomonas titanicae KHS3 encodes a methyltransferase specific to its cognate receptor

RAMOS RICCIUTI, F.E.; STUDDERT, C.A.; HERRERA SEITZ, M. K.

Salta

Congreso; LV Reunión de Sociedad Argentina de Investigaciones Bioquímicas (SAIB); 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The environmental strain Halomonas titanicae KHS3, isolated from Mar del Plata harbor, encodes two chemosensory systems. The canonical che cluster, che1, contains all the genes needed to control the flagellar movement in response to chemical gradients. The second cluster, che2, is homologous to the wsp cluster from Pseudomonas that has been implicated in biofilm formation. Che2 consists of seven putative proteins: a chemoreceptor Htc10, a methyltransferase CheR2, an esterase CheB2, a histidine kinase CheA2, two CheW-like coupling proteins and a diguanylate cyclase. We hypothesize that Htc10 transmits information from the extracellular medium to CheA2, through CheW protein/s. This kinase activates the diguanylate cyclase by phosphorylation, affecting the levels of cyclic di-GMP and, consequently, some downstream process/es. The aim of this work was to investigate the specificity of CheR2 as a methyltransferase. To assess whether CheR2 was able to methylate the chemoreceptor Htc10, both proteins were expressed in E. coli cells. The presence of CheR2 caused a shift in the electrophoretic mobility of Htc10, indicating that it was indeed methylated. The occurrence of Htc10 methylation by CheR2 was confirmed by mass spectrometry. MS results revealed methylation in conserved glutamate residues, within a region that corresponds to the adaptation domain of canonical chemoreceptors. In contrast, CheR2 did not change the mobility of Tsr, the serine E. coli chemoreceptor. Likewise, the methyltransferase CheR from E. coli changed the mobility of Tsr but not that of Htc10, indicating that both methyltransferases showed substrate specificity. In addition to the methyltransferase domain, CheR2 has tetratricopeptide repeats (TPR), absent in E. coli CheR. To assess whether the TPR domain affected CheR2 activity, we created a truncated protein, through site directed mutagenesis, with a stop codon before TPR. In SDS-PAGE, the patterns of Htc10 bands in the presence of full-length or truncated CheR2 were indistinguishable, indicating that both forms of the enzyme were capable of Htc10 modification. To find out the role of the che2 cluster in Halomonas, we introduced an expression vector encoding CheR2 in wild-type Halomonas titanicae. Since the unbalanced expression of the methyltransferase is expected to shift the population of Htc10 towards a more methylated state and perhaps affect the downstream processes controlled by this system, we analyzed biofilm formation, motility and cell adherence in transformed Halomonas under induced conditions. So far we did not detect significant changes in the analyzed behaviors. We have demonstrated that CheR2 functions as a dedicated methyltransferase that uses Htc10 as a substrate but not a canonical chemotaxis receptor. On-going studies are directed towards the achievement of higher overexpression systems to assess a variety of phenotypes. This might help us to find clues about the main role of this chemosensory system.