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23 Congress of the International Union for Biochemistry and Molecular Biology
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44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
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Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
PhaP1 IS THE MAIN PHASIN CONTROLLING PHB ACCUMULATION IN
Herbaspirillum seropedicae SmR1
Luis Paulo Silveira Alves1, Evandro Tirapelle1, Emanuel Maltempi de Souza1,
Fabio de Oliveira Pedrosa1, Leda Satie Chubatsu1, Marcelo Müller-Santos1
1
Departamento de Bioquímica, Núcleo de Fixação de Nitrogênio,
Universidade Federal do Paraná, Curitiba, Brazil.
Abstract
Introduction. Polyhydroxybutyrate (PHB) is a polymer stored by several bacteria
as intracellular granules. On the surface of PHB granules, there is a proteinaceous
layer composed mainly by phasins. Phasins control the number and size of PHB
granules and are important for granules stability. H. seropedicae has the phasins
PhaP1 and PhaP2, sharing 65% identity. Objective. In this work, we have
characterized H. seropedicae mutants defective in the expression of phaP1,
phaP2 or both genes. Materials and methods. To construct the mutants, a DNA
fragment carrying 500 bp from upstream and downstream regions of the phaP
genes were cloned into pK18mobsacB. The resulting plasmids were transferred to
H. seropedicae by mating with E. coli S17-1. Double cross-over mutants carrying
the deletions were selected in medium containing 10% of sucrose.
Complementation was obtained with plasmids carrying phaP1 or phaP2 under
control of their native promoters. Expression of phaP genes was also evaluated
through beta-galactosidase activity in strains harboring fusions of the 5’-UTR
regions of phaP genes with promoter-less lacZ. The PHB production was
measured by methanolysis of lyophilized cells followed by gas chromatography
analysis. Results. Despite of high identity between PhaP1 and PhaP2, our results
indicate that PhaP1 is the main phasin in H. seropedicae, since its deletion
reduced PHB accumulation by 50% in comparison to the parental strains. PhaP2
partially compensates for the absence of PhaP1, maintaining 50 % of the parental
PHB level. PHB accumulation was fully impaired in the absence of both phasins.
The expression of phasins was differentially regulated, wherein phaP2 was highly
expressed only when phaP1 was deleted. Conclusion. PhaP1 is the main phasin
for PHB accumulation in H. seropedicae, but PhaP2 partially compensates its
absence. The profile of expression indicates that PhaP2 can act as a backup
phasin to maintain PHB production in H. seropedicae.
Acknowledgements: INCT-Fixação Biológica de Nitrogênio, CNPq, Fundação
Araucária, CAPES.
Key words: phasin, polyhydroxybutyrate (PHB), Herbaspirillum seropedicae.
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)