Cloning, expression and characterization of two chemoreceptors from Halomonas sp KHS3

PROSA L. ; GASPEROTTI A. F. ; STUDDERT C. A. ; HERRERA SEITZ M. K.

Mar del Plata

Congreso; 51st Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2015

SAIB

Halomonas sp KHS3 was isolated from a hydrocarbon contaminated area from Mar del Plata harbor. This strain is able to grow on aromatic hydrocarbons and exhibits chemotactic responses towards these compounds. The analysis of its whole genomic sequence showed the presence of twenty-four chemoreceptors (MCPs), including at least one that is presumably involved in the specific responses to hydrocarbons. Based on their genomic context, two MCPs were chosen for cloning and expression in E. coli cells, named PEG_925 and PEG_4119. PEG_925 is located within acluster of chemotaxis genes, and is possibly involved in general nutrient chemotaxis response, while PEG_4119 is found in a cluster containing genes of the salicylate pathway. Flagellar rotation assays in E. coli cells expressingPEG_925 or PEG_4119 indicated that they are able to activate and control the kinase CheA. Using the same assay, we explored possible ligands and found that PEG_925 responds to aspartate as an attractant. PEG_4119, on the other hand, responded to lactic acid as an attractant. Higher order organization was analyzed through subcelullar localization assays using YFP fusion proteins and crosslinking assays, indicating a proper interaction between these MCPs with other components of the chemotactic signaling complex of E. coli