Abstract
Phasins are important proteins controlling poly-3-hydroxybutyrate (PHB) granules formation, their number into the cell and stability. The genome sequencing of the endophytic and diazotrophic bacterium Herbaspirillum seropedicae SmR1 revealed two homologous phasin genes. To verify the role of the phasins on PHB accumulation in the parental strain H. seropedicae SmR1, isogenic strains defective in the expression of phaP1, phaP2 or both genes were obtained by gene deletion and characterized in this work. Despite of the high sequence similarity between PhaP1 and PhaP2, PhaP1 is the major phasin in H. seropedicae, since its deletion reduced PHB accumulation by ≈50% in comparison to the parental and ΔphaP2. Upon deletion of phaP1, the expression of phaP2 was sixfold enhanced in the ΔphaP1 strain. The responsive backup expression of phaP2 partially rescued the ΔphaP1 mutant, maintaining about 50% of the parental PHB level. The double mutant ΔphaP1.2 did not accumulate PHB in any growth stage and showed a severe reduction of growth when glucose was the carbon source, a clear demonstration of negative impact in the fitness. The co-occurrence of phaP1 and phaP2 homologous in bacteria relatives of H. seropedicae, including other endophytes, indicates that the mechanism of phasin compensation by phaP2 expression may be operating in other organisms, showing that PHB metabolism is a key factor to adaptation and efficiency of endophytic bacteria.
Highlights
Herbaspirillum seropedicae SmR1 is a diazotrophic β-Proteobacterium that associates beneficially with economically relevant species of Gramineae (Baldani et al, 1986) and produces poly-3hydroxybutyrate (PHB) granules as means of carbon and energy storage (Catalan et al, 2007; Kadowaki et al, 2011)
The phylogenetic analysis of PhaP1 and PhaP2 homolog sequences showed that the appearance of a second phasin occurred early in evolution of the Herbaspirillum genus and was conserved in subsequent speciation events (Figure 1E), indicating that the presence of both phasins should be important for PHB accumulation
To analyze the role of phasins in H. seropedicae, we correlated their presence with PHB production and expression of phaP1 and phaP2
Summary
Herbaspirillum seropedicae SmR1 is a diazotrophic β-Proteobacterium that associates beneficially with economically relevant species of Gramineae (Baldani et al, 1986) and produces poly-3hydroxybutyrate (PHB) granules as means of carbon and energy storage (Catalan et al, 2007; Kadowaki et al, 2011). Thirteen genes probably involved in PHB metabolism were identified in the strain SmR1 (Kadowaki et al, 2011; Pedrosa et al, 2011), including four phaC, two phaZ and two phaP genes encoding PHA synthases, PHA depolymerases and phasins, respectively. H. seropedicae SmR1 possesses three genes encoding proteins homologous to PHA synthases (Pedrosa et al, 2011), PHB synthesis is supported by the PHA synthase expressed by phaC1, since its deletion abolishes PHB accumulation (Tirapelle et al, 2013). PHB plays important roles in nitrogen fixation and plant–bacteria interactions (Mandon et al, 1998; Kadouri et al, 2003; Wang et al, 2007; Quelas et al, 2013), but its action in H. seropedicae was not been fully characterized
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