Uma nova proteína autotransportadora e sua influência no swarming e na produção de raminolipídeos em Burkholderia thailandensis

Abstract

Burkholderia thailandensis (Bt) is a non-infectious species, commonly isolated from soil and water. It has been widely used as a surrogate model for studying many characteristics of its pathogenic counterparts within Burkholderia spp., which include the ability to produce rhamnolipids, and are directly involved in multicellular behavior. The study of rhamnolipids’ biosynthetic pathways in Pseudomonas aeruginosa revealed an autotransporter protein called EstA, which greatly influences the production of rhamnolipids, swarming motility, and the formation of adherent biofilms. Our group has since focused on Bt to investigate the presence of autotransporter proteins and their phenotypes, which led to the identification of an EstA-like autotransporter gene (herein designated orfE264). Therefore, we hypothesized that the protein encoded by orfE264 could play similar roles to those of EstA in Bt’s biosurfactant production and related phenotypes. We used the wild-type Bt E264 and its orfE264-defficient derivatives for functional assays. The swarming motility was performed in M9 medium supplemented with 0,5% agarose, revealing a significantly decreased swarming motility and rhamnolipid production when compared to the wild-type strain, strongly suggesting that this gene is involved in those phenotypes. The direct correlation between the swarming surface and the production levels of rhamnolipids suggests that OrfE264 plays a pivotal role in biosynthesis and/or secretion of this biomolecule. On the other hand, in trans complementation of the orfE264-knockout strain led to an increased production of rhamnolipids and swarming motility. Interestingly, cross-complementation of the orfE264-mutant with full-length estA failed to restore the phenotypes. To determine the subcellular localization of OrfE264, outer-membrane vesicles (OMVs) and the membrane fraction of an E. coli LMG 194 with a plasmid containing the recombinant orfE264 gene were isolated and analyzed by fluorescence enzymatic assays and mass spectrometry, respectively. These results showed that OrfE264 is an outer-membrane protein with an active esterase domain. The use of B. thailandensis for industrial production of rhamnolipids represents a great interest in biotechnology, and an EstA-like autotransporter might serve as a suitable target for metabolic engineering, aiming to reduce the production costs for this biosurfactant.