Caracterización de las interacciones de la fasina PhaF en la bacteria modelo acumuladora de polihidroxialcanoatos, Pseudomonas putida KT2440

The discovery of bacterial polyhydroxyalkanoates (PHA) in the cytoplasm of Bacillus megaterium by Lemoigne (Lemoigne, 1926) unveiled a new cla ss of in vivo synthesized polymers with adjustable properties and different applications (as plastics, in medical devices, etc.); it also provided impetus to research on proteins involved in PHA accumulation. Interest in phasins emerged through two major observations: i) that they bind to PHA, potentially establishing a network-like protein layer on the surface of intracellular PHA granules; and ii) that their concentration can reach 5% (wt/wt) of the total protein content of PHA-accumulating cells (Wieczorek et al., 1995; Maestro and Sanz, 2017). Soon after it was found that these proteins are almost exclusively produced when PHA is synthesized (York et al., 2002), and that they play important roles in its biogenesis, helping to ensure optimal cell fitness. For example, phasins have been found to engage in the physical stabilization of PHA granules at the polymer-cytoplasm interface, and to participate in the control of the number and size of these granules, their segregation into daughter cells, and their mobilization (reviewed in (Mezzina and Pettinari, 2016; Maestro and Sanz, 2017)). Although these proteins are highly diverse in sequence, they share structural features such as a majority amphipathic α-helix composition, and all show intrinsic disorder in absence of PHA. Structural models have additionally revealed them to possess coiled-coil regions that might be important in the establishment of protein-protein interactions (Maestro et al., 2013). However, experimental evidence of coiled-coil mediated oligomerization in these proteins has been lacking. In the first chapter of this PhD thesis, structure prediction analyses are used to characterize the coiled-coil domains of phasins PhaF and PhaI produced by the model PHA-accumulating bacterium Pseudomonas putida KT2440. Their oligomeriza tion is examined using biolayer interferometry and the in vivo two-hybrid (BACTH) system. The interaction capacity of a series of coiled-coil mutant phasins is also explored. The results confirm the formation of PhaF and PhaI complexes, and establish the involvement of a predicted short leucine zipper-like coiled-coil (ZIP), which contains “ideal” residues located within the hydrophobic core, in the oligomer's stability. The substitution of key residues (leucines or valines) in PhaI ZIP (ZIPI) for alanine is shown to reduce oligomerization efficiency by around 75%. These results indicate that coiled-coil motifs are essential in PhaF-PhaI interactions, and that the correct oligomerization of the phasins requires the formation of a stable hydrophobic interface between them. Since this motif is present in most phasins produced by PHA-accumulating bacteria, the present results provide valuable insights into how PHA granule stability might be modified to fulfil diverse applications. In spite of the high sequence similarity shared by PhaF and PhaI, the deletion of phaF is w ell know n to ha ve a nota ble impact on overa ll cell physiology, resulting in a non-homogeneous population in terms of PHA content and granule localization, and in the reduction of the expression of phaC1 (PHA synthase) and phaI (Prieto et al., 1999; Galan et al., 2011; Dinjaski and Prieto, 2013). This is most likely due to the presence of the C-terminal or DNA binding domain in PhaF, which is absent in PhaI. Taking into account this observations, and the protein concentration at the surface of the granule, it was hypothesized in this work that PhaF might participate in extensive networks of protein–protein interactions that assemble and disassemble in obedience to specific cellular signals. The second chapter discusses the comprehensive approach followed to identify candidates for novel interactions of PhaF. Mass spectrometry-coupled recombinant His6-PhaF interaction/pull-down (affinity chromatography) experiments were used to screen for P. putida KT2440 proteins that interact with PhaF. Six were identified as forming complexes with PhaF, including two PHA-related proteins: phasin PhaI (UniProt Q88D20), and the transcriptional regulator PhaD (Q88D22). When these results were correlated with PHA granule proteome results, also accomplished in this Thesis, four of the latter six proteins were again identified. The most interesting finding, however, was the interaction between the PHA regulator protein PhaD and PhaF in the pull-down assay. PhaD functions as an activator of phaC1 and phaI transcription. It binds to 25 and 29 bp target regions of the PC1 and PI promoters respectively (which lie upstream of phaC1 a nd phaI genes) (de Eugenio et a l., 2010). Rema rka bly, the a bsence of Pha D in the PHA granules, corroborated by in vivo localization of a PhaD-GFP fusion protein, suggests the existence of a novel mechanism in which the PhaD-PhaF complex participates in the regulatory system controlling pha genes expression. In vivo BACTH (tw o-hybrid) interaction assays confirmed the interaction of PhaF and PhaD. Electrophoretic mobility shift analysis of the phaI promoter revealed the formation of a DNA-PhaD-PhaF complex that migrates to a position on the gel different to those reached when the PI DNA fragment is complexed with the two proteins individually. Based on these results, it might be concluded that PhaF and PhaD act in harmony to control the expression of pha genes. The disclosure of in vivo roles for phasins over recent years has opened up a new field of research which holds the promise of developing innovative applications based on these proteins. The strong affinity of phasins for PHA (and other hydrophobic materials) has already led to the development of affinity tags for use in low-cost recombinant protein purification, specific drug delivery vehicles, and cell sorting techniques. Additionally, their amphiphilic nature means phasins have uses as biosurfactants (Wei et al., 2011) and the functionalization of biomaterials through coating their surfaces in combination with cell signal molecules (Dong et al., 2010; Xie et al., 2013; Gao et al., 2014). Despite the remarkable properties of phasins, and their recently explored use in the surface coating of biomaterials, we know little about their robustness and stability at interfaces, or about their adsorption behavior with respect to PHA (their natural substrates). Neither has the alteration of the molecular conformation of either during the functionalization process been addressed. The Langmuir technique was therefore used to study the interaction of PhaF with PHA by mimicking the native environment, thus providing insights into the structure of the PHA-PhaF complex. The results could help to improve control over phasin-coated material production. The Langmuir technique, combined with in situ microscopy and spectroscopic methods, revealed PhaF to form stable and robust monolayers at different temperatures, with an almost flat orientation of its alpha-helix at the air-water interface. The adsorption of PhaF onto preformed PHA monolayers yielded stable mixed-layers below a surface pressure of π = ~15.7 mN/m. Further PhaF adsorption induced a molecular reorganization of the film. In the prospective of the assumption made in this work, the surface of granules being formed merely by proteins, these results let us conclude that elevated concentrations of PhaF function as a surfactant, separating PHA into “granules” in hydrophilic environments as occurs in the cytoplasm of bacterial cells. Furthermore, PHA polymers with stronger surface hydrophobicity are here shown to be more appropriate substrates for PhaF-mediated functionalization than less hydrophobic polyesters like PLGA, poly[(lactide-co-glycolide)]. The observed orientation of the main axis of the protein in relation to both polyester surfaces ensures the best exposure of the hydrophobic residues. A suitable coating strategy for producing PHA-functionalized materials is thus unveiled. The Langmuir technique also allowed for the preparation of monomolecular films of PhaF that were successfully transferred to solid substrates and characterized by atomic force microscopy (AFM). This chapter presents the first thorough analysis of the surface properties of PhaF, and the results obtained help to improve our understanding of already used PHA-based biomaterials, and perhaps other polyester-based biomaterials.