Suboptimal expression of the Lon B-protease increases bacterioruberin and lipid content in the haloarchaeon Haloferax volcanii.

CERLETTI, M.; GIMÉNEZ, M.; DE CASTRO, R

Rosario (Santa Fe)

Congreso; IX Congreso de Microbiología General SAMIGE; 2013

SAMIGE

Archaea were classified as a separate domain from Bacteria and Eukarya due to their evolutionary origin and distinct molecular features. Many of the representative members are extremophiles, thus, these unusual microbes are a valuable resource for basic and applied science. Proteolysis is a key process in cell physiology, however, current knowledge on protease function and regulation in archaea is limited. ATP-dependent Lon proteases are ubiquitous in the three domains of Life. While the soluble Lon proteases of bacteria and eukaryotic organelles (LonA) have been deeply characterized, the role of the archaeal-type enzyme (LonB) in the physiology of archaea remains unknown. We previously constructed a lon deletion mutant in the haloarchaeon Haloferax volcanii (Hv). This strain was successfully obtained when a copy of the wt gene was provided in trans, suggesting that Lon was essential for viability in this microorganism. Considering the lethal phenotype of the lon mutation, the aim of this study was to construct and characterize a conditional mutant of the Hvlon gene. This strain (HvH26 ptnaA-lon-abi) was generated by inserting the trp-regulatable promoter ptnaA of H. volcanii upstream the lon sequence. Additionally, a deletion mutant in the downstream gene abi was constructed (HvH26 ∆abi) to evaluate the potential contribution of this gene product. Suboptimal levels of Lon protease severely affected cell growth as well as increased bacterioruberin (cell pigmentation) and lipid content. On the contrary, the HvH26 ∆abi mutant remained unchanged, indicating that the differential phenotypes evidenced by the conditional mutant were due to suboptimal Lon expression. Deficiency in the Lon protease also produced hypersensitivity to puromycin (a protein synthesis inhibitor) and to lovastatin (an inhibitor of the HMG-CoA reductase, a key enzyme in isoprenoid biosynthesis). These results suggest that Lon participates in protein quality control, and that it may be involved in the regulation of isoprenoid metabolism. The inability to dispose defective peptides and/or control an adequate lipid composition of the cell membrane most likely accounts for loss of viability in H. volcanii lacking this protease.