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7 páginas, 9 figuras -- PAGS nros. 402-418, The PhaF is a nucleoid-associated like protein of Pseudomonas putida KT2442 involved in the polyhydroxyalkanoate (PHA) metabolism. Its primary structure shows two modular domains; the N-terminal PHA granule-binding domain (phasin domain) and the C-terminal half containing AAKP-like tandem repeats characteristic of the histone H1 family. Although the PhaF binding to PHA granules and its role as transcriptional regulator have been previously demonstrated, the cell physiology meaning of these properties remains unknown. This work demonstrates that PhaF plays a crucial role in granule localization within the cell. TEM and flow cytometry studies of cells producing granules at early growth stage demonstrated that PhaF directs the PHA granules to the centre of the cells, forming a characteristic needle array. Our studies demonstrated the existence of two markedly different cell populations in the strain lacking PhaF protein, i.e. cells with and without PHA. Complementation studies definitively demonstrated a key role of PhaF in granule segregation during the cell division ensuring the equal distribution of granules between daughter cells. In vitro studies showed that PhaF binds DNA through its C-terminal domain in a non-specific manner. All these findings suggested a main role of PhaF in PHA apparatus through interactions with the segregating chromosome, This work was supported by grants from the Comunidad Autónoma de Madrid (P-AMB-259-0505), the Comisión Interministerial de Ciencia y Tecnología (BIO2007-67304-C02, BIO2010-21049, CSD2007-00005) and by European Union Grants (GEN 2006-27750-C5-3-E and NMP2-CT-2007-026515)

Narrow-host-range plasmid pPS10, originally found in Pseudomonas savastanoi, is unable to replicate in other strains such as Escherichia coli. Here, we report that the establishment of pPS10 in E. coli can be achieved by a triple mutation in the dnaA gene of E. coli (dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant). As the E. coli strain used contained double amber suppressor mutations (supE, supF), the amber codon in dnaA403 can be translated into glutamine or tyrosine. Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon in E. coli. The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity. To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon in E. coli and provides support for the hypothesis that interactions between RepA and DnaA modulate the establish-ment of pPS10 in that bacteria and probably in other species., B.M. was supported by an FPI predoctoral fellowship from the Spanish Ministerio de Educación y Ciencia and a short-term EMBO fellowship. This work was supported by Contrato-Programa de la CAM Grupos Estratégicos 2000–2003, grants BIO98-0106 and QL K2-CT-2000-00634 from the EC and grant BIO99-0859-CO1 from the Spanish MEC.

14 p.-9 fig.-4 tab., The ββ-solenoid structures are part of many proteins involved in the recognition of bacterial cell wall. They are elongated polypeptides consisting of repeated β-hairpins connected by linker sequences and disposed around a superhelical axis stabilised by short-range interactions. Among the most studied ββ-solenoids are those belonging to the family of choline-binding modules (CBMs) from the respiratory pathogen Streptococcus pneumoniae (pneumococcus) and its bacteriophages, and their properties have been employed to develop several biotechnological and biomedical tools. We have carried out a theoretical, spectroscopic and thermodynamic study of the ββ-solenoid structure of the CBM from the pneumococcal LytA autolysin using peptides of increasing length containing 1–3 repeats of this structure. Our results show that hints of native-like tertiary structure are only observed with a minimum of three β-hairpins, corresponding to one turn of the solenoid superhelix, and identify the linker sequences between hairpins as the major directors of the solenoid folding. This study paves the way for the rational structural engineering of ββ-solenoids aimed to find novel applications., Funding sources: grants BIO2016-79323-R, CTQ2017-84371P and PID2019-105126RB-I00 (Agencia Estatal de Investigacion AEI/FEDER-EU-10.13039/ 501100011033-, and Ministerio de Ciencia e Innovación, Spain) and Intramural incorporation project #201820I132 (CSIC, Spain).

41 p.-4 fig.-5 tab., Bacterial biopolymers are naturally occurring materials comprising a wide range of molecules with diverse chemical structures that can be produced from renewable sources following the principles of the circular economy. Over the last decades, they have gained substantial interest in the biomedical field as drug nanocarriers, implantable material coatings, and tissue-regeneration scaffolds or membranes due to their inherent biocompatibility, biodegradability into nonhazardous disintegration products, and their mechanical properties, which are similar to those of human tissues. The present review focuses upon three technologically advanced bacterial biopolymers, namely, bacterial cellulose (BC), polyhydroxyalkanoates (PHA), and γ-polyglutamic acid (PGA), as models of different carbon-backbone structures (polysaccharides, polyesters, and polyamides) produced by bacteria that are suitable for biomedical applications in nanoscale systems. This selection models evidence of the wide versatility of microorganisms to generate biopolymers by diverse metabolic strategies. We highlight the suitability for applied sustainable bioprocesses for the production of BC, PHA, and PGA based on renewable carbon sources and the singularity of each process driven by bacterial machinery. The inherent properties of each polymer can be fine-tuned by means of chemical and biotechnological approaches, such as metabolic engineering and peptide functionalization, to further expand their structural diversity and their applicability as nanomaterials in biomedicine., The authors thank the Community of Madrid [P2018/NMT4389], the Spanish Ministry of Science, Innovation, and Universities [BiO2017-8344-8-R, PID2019-105126RB-I00], and the European Union’s Horizon 2020 Research and Innovation Programme [grant agreement no 870294 (Mix-Up)] for the financial support of this project. Francisco Blanco is the recipient of a predoctoral grant from the State Program for the Promotion of Talent and Its Employability in R&D&I (PRE-2018-083859) from the Spanish Ministry of Science and Innovation.

26 p.-6 fig.-3 tab., Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens., This work was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness (BIO2013-47684-R, BIO2016-79323-R and SAF2017-88664-R), and the RETICS-FEDER RICET RD16/0027/0010.

13 p.-7 fig., Advanced biomaterials provide an interesting and versatile platform to implement new and more effective strategies to fight bacterial infections. Chitosan is one of these biopolymers and possesses relevant features for biomedical applications. Here we synthesized nanoparticles of chitosan derivatized with diethylaminoethyl groups (ChiDENPs) to emulate the choline residues in the pneumococcal cell wall and act as ligands for choline-binding proteins (CBPs). Firstly, we assessed the ability of diethylaminoethyl (DEAE) to sequester the CBPs present in the bacterial surface, thus promoting chain formation. Secondly, the CBP-binding ability of ChiDENPs was purposed to encapsulate a bio-active molecule, the antimicrobial enzyme Cpl-711 (ChiDENPs-711), with improved stability over non-derivatized chitosan. The enzyme-loaded system released more than 90% of the active enzybiotic in ≈ 2 h, above the usual in vivo half-life of this kind of enzymes. Therefore, ChiDENPs provide a promising platform for the controlled release of CBP-enzybiotics in biological contexts., This study was funded by a grant from the Ministerio de Economía y Competitividad (MINECO-FEDER, SAF2017-88664-R) and the Ministerio de Ciencia e Innovación (MAT2017-84277-R, PID2019-105126RB-I00). F. J. Caro acknowledges financial support from CONACyT (Mexico) through the scholarship ‘Apoyo para estancias postdoctorales en el extranjero vinculadas a la consolidación de grupos de investigación y fortalecimiento del Posgrado nacional’. Additional funding was provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) and Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), an initiative of the Instituto de Salud Carlos III.

22 p.-9 fig.-3 tab., This work was supported by funding from the European Union's Horizon 2020 research and innovation program under grant agreement numbers 633962 (P4S project) and 814418 (SinFonia project). We also acknowledge support from the Community of Madrid (P218/NMT4389), Instituto de Saluid Carlos III (CIBER de enfermedades respiratorias), and the Spanish Ministry of Science, Innovation and Universities (BIO2016-79323-R, BIO2017-83448_R).

52 p.-9 fig.-3 tab., Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for the binding to the PHA granule. BioF has been exploited with biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we have analysed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain the affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on the PHA, stability on the surface of the PHA granule against pH, temperature and ionic strength, and their possible influence on the PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP), which retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing functionality and stability of the fusion proteins MinP-β-galactosidase and MinP-CueO from Escherichia coli., IMPORTANCE.Polyhydroxyalkanoates (PHAs) are biocompatible, non-toxic and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67 % shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo., This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme, grant agreement no. 633962 (P4SB), the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R, BIO2016-79323-R, RTI2018-094564-B-I00) and the Community of Madrid (P2013/MIT2807, P2018/NMT4389). Francisco Blanco is a recipient of a predoctoral grant from the State 560 Program for the Promotion of Talent and its Employability in R&D&I (PRE-2018- 561 083859) from the Spanish Ministry of Science and Innovation.

38 p.-6 fig.-4 fig. supl.-2 tab. supl., Background Phasins are low molecular mass proteins that accumulate strongly in bacterial cells in response to the intracellular storage of polyhydroxyalkanoates (PHAs). Although lacking catalytic activity, phasins are the major components of the surface of the PHA granules and could be potentially involved in the formation of a network-like protein layer surrounding the polyester inclusions. Structural models revealed phasins to possess coiled-coil regions that might be important in the establishment of protein-protein interactions. However, there is not experimental evidence of a coiled-coil mediated oligomerization in these proteins., Methods Structure prediction analyses were used to characterize the coiled-coil motifs of phasins PhaF and PhaI –produced by the model bacterium Pseudomonas putida KT2440-. Their oligomerization was evaluated by biolayer interferometry and the in vivo two-hybrid (BACTH) system. The interaction ability of a series of coiled-coil mutated derivatives was also measured., Results The formation of PhaF and PhaI complexes was detected. A predicted short leucine zipper-like coiled-coil (ZIP), containing “ideal” residues located within the hydrophobic core, was shown responsible for the oligomers stability. The substitution of key residues (leucines or valines) in PhaI ZIP (ZIPI) for alanine reduced by four fold the oligomerization efficiency., Conclusions These results indicate that coiled-coil motifs are essential for phasin interactions. Correct oligomerization requires the formation of a stable hydrophobic interface between both phasins., General Significance Our findings elucidate the oligomerization motif of PhaF and PhaI. This motif is present in most phasins from PHA-accumulating bacteria and offers a potentially important target for modulating the PHA granules stability., This work was supported by the European Union’s Horizon 2020 research and innovation program (nº 633962), the Community of Madrid (P2013/MIT2807), and the Spanish Ministry of the Economy (BIO2017-83448-R and BIO2016-79323-R).

12 p.-6 fig.-2 tab., The presence of synthetic dyes in wastewaters generated by the textile industry constitutes a serious environmental and health problem that urges the scientific community on an appropriate action. As a proof-of-concept, we have developed a novel approach to design enzymatic bioreactors with the ability to decolorize dye solutions through the immobilization of the bacterial CueO laccase-like multicopper oxidase from Escherichia coli on polyhydroxybutyrate (PHB) beads by making use of the BioF affinity tag. The decolorization efficiency of the system was characterized by a series of parameters, namely maximum enzyme adsorption capacity, pH profile, kinetic constants, substrate range, temperature and bioreactor recycling. Depending on the tested dye, immobilization increased the catalytic activity of CueO by up to 40-fold with respect to the soluble enzyme, reaching decolorization efficiencies of 45-90%. Our results indicate that oxidase bioreactors based on polyhydroxyalkanoates are a promising alternative for the treatment of coloured industrial wastewaters., This work was supported by Spanish Ministry of Economy and Competitiveness (grants BIO2013-47684-R and BIO2016-79323-R),IVACE (Institut Valenciá de Competitivitat Empresarial, SPAIN) and FEDER (Fondo Europeo de Desarrollo Regional, Europe).