PROTECTIVE EFFECT OF PHASINS PhaP FROM Azotobacter sp.FA-8 AND PhbP FROM Pseudomonas extremaustralis IN RECOMBINANT Escherichia coli

DANIELA ÁLVAREZ; MARIELA P. MEZZINA; M. JULIA PETTINARI

Córdoba

Congreso; XI Congreso Argentino de Microbiología General (SAMIGE); 2015

Sociedad Argentina de Microbiología General (SAMIGE)

Phasins are proteins associated to intracellular polyhydroxyalkanoate (PHA) granules, a biodegradable polymer accumulated by many bacteria under unfavorable conditions as a carbon and energy reserve. Phasins not only play an important structural role in the polymer accumulation and the number and size of the granules, but have also been shown to have regulatory functions. It has been reported that PHA accumulation in recombinant E.coli strains causes stress in cells, evidenced in the increased expression of stress-related proteins such as chaperones IbpA and DnaK. This suggests that PHA production induces the heat shock response by titrating available cytoplasmic chaperones onto the surface of the PHA granules. The presence of PhaPAz in these PHA-accumulating strains exhibited a decrease in the heat shock response and, consequently, the expression of these chaperones. Furthermore, PhaPAz expression also has a protective effect in non-PHA-producing E. coli strains, evidenced in the increased growth and higher resistance to heat shock and superoxide stress. The effect described for this phasin is not only passive but involves an active role in promoting protein folding, and preventing unfolding due to its chaperone-like function. The aim of this work is to analyze if the protective effect observed for PhaPAz is a common feature among phasins. To help elucidate this, we studied the effect of the expression of phasin PhbP from Pseudomonas extremoaustralis (PhbPPe ) in recombinant E. coli. An in silico comparative analysis of PhaPAz and PhbPPe revealed that both phasins have similar secondary structures, consisting of α-helices and random coil regions. phbP was amplified by PCR from P. extremoaustralis genomic DNA and cloned in a plasmid that allowed overexpression in E. coli. The protective effect of this phasin was studied by analyzing the level of expression of the small heat shock protein IbpA, which is a stress indicator. For this purpose, strain ADA100, which carries a fusion of lacZ to the ibpA promoter in the chromosome, was transformed with this plasmid in order to analyze ibpA expression levels by the β-galactosidase activity assay, in normal and heat stress conditions. In addition, resistance to heat shock was also studied by determining cell viability after incubation at 52ºC, by the colony count method. As previously observed for PhaPAz, PhbPPe was also shown to protect E. coli from heat shock. If the effects described for PhaPAz are a common feature among phasins, these proteins could play an important role in protein homeostasis both in natural and recombinant PHA producers, complementing the resources provided from polymer degradation with specific chaperone activities that could protect the cells from different types of stress.