STABILIZATION OF PHYTOENE SYNTHASE AFFECTS GROWTH RATE BUT DOES NOT ACCOUNT FOR THE LETHALITY OF THE lonB MUTATION IN THE ARCHAEON Haloferax volcanii

DE CASTRO, R.E; CERLETTI, M.; FERRARI, M.C.

San Miguel de Tucuman

Congreso; XII Congreso Argentino de Microbiología General; 2017

Sociedad Argentina de Microbiología General

Haloarchaea thrive in hypersaline lakes and solar salterns (> 2 M NaCl). Most strains display pink/red coloniesdue to the presence of carotenoid pigments (mainly bacterioruberins) that protect the cells fromUV light irradiation. Energy-dependent proteolysis is a key process in cell physiology as it maintains thequality of proteins and regulates numerous cellular functions. The ATP-dependent Lon protease is conservedamong the three domains of Life, however, its biological relevance in archaeal cells has not beenelucidated. We have previously investigated the function of LonB in a conditional lonB mutant of thehaloarchaeon Haloferax volcanii (HVLON3) and concluded that this protease is essential for cell viability;moreover, suboptimal Lon levels affected the growth rate, cell shape and produced hyperpigmentation.Proteomic analysis showed that LonB deficiency causes a dramatic accumulation of phytoene synthase(PSY), the key enzyme in carotenoid biosynthesis and that PSY degradation is LonB-dependent. Theincrease in carotenoids content likely affects the functionality of the cell membrane which is reflectedby a decrease in growth rate in HVLON3 and may be responsible for the lethality of the lon mutation.To test this hypothesis, in this work we constructed a H. volcanii strain with regulatable PSY levels andobserved that overexpression of this enzyme led to increased bacterioruberins cell content (4-fold) witha concomitant decrease in growth rate (μ 0.075 vs 0.033 h-1 ). To investigate whether stabilizationof PSY impaired viability of lon KO mutants in H. volcanii, we attempted to delete the lon gene froma deltacrtB (PSY) background. We were unable to detect clones that had completely deleted the longene, suggesting that PSY stabilization is not the sole/main reason for the indispensability of LonB inH. volcanii. On the other hand, the crtB gene was deleted from the chromosome of HVLON3 renderingalmost all the resulting clones unpigmented. The high efficiency of psy KO mutation (nearly 100%) inaddition to the fact that these mutants showed a higher growth rate than the parental strain HVLON3suggests that even though PSY stabilization may not be the sole reason for the lethality of the lon mutation,it severely affects H. volcanii growth performance. Key words: Archaea, Lon protease, phytoenesynthase. Ferrari and Cerletti contributed equally to this work Supported by CONICET, UNMdP, ANPCyTand MINCyT-BMBF.