Nature-inspired material binding peptides with versatile polyester affinities and binding strengths.

Biodegradable polyesters, such as polyhydroxyalkanoates (PHAs), are having a tremendous impact on biomedicine. However, these polymers lack functional moieties to impart functions like targeted delivery of molecules. Inspired by native GAPs, such as phasins and their polymer-binding and surfactant properties, we generated small material binding peptides (MBPs) for polyester surface functionalization using a rational approach based on amphiphilicity. Here, two peptides of 48 amino acids derived from phasins PhaF and PhaI from Pseudomonas putida, MinP and the novel-designed MinI, were assessed for their binding towards two types of PHAs, PHB and PHOH. In vivo, fluorescence studies revealed selective binding towards PHOH, whilst in vitro binding experiments using the Langmuir-Blodgett technique coupled to ellipsometry showed KD in the range of nM for all polymers and MBPs. Marked morphological changes of the polymer surface upon peptide adsorption were shown by BAM and AFM for PHOH. Moreover, both MBPs were successfully used to immobilize cargo proteins on the polymer surfaces. Altogether, this work shows that by redesigning the amphiphilicity of phasins, a high affinity but lower specificity to polyesters can be achieved in vitro. Furthermore, the MBPs demonstrated binding to PET, showing potential to bind cargo molecules also to synthetic polyesters.

Enzybiotic-mediated antimicrobial functionalization of polyhydroxyalkanoates.

Polymeric nanoparticles (NPs) present some ideal properties as biomedical nanocarriers for targeted drug delivery such as enhanced translocation through body barriers. Biopolymers, such as polyhydroxyalkanoates (PHAs) are gaining attention as nanocarrier biomaterials due to their inherent biocompatibility, biodegradability, and ability to be vehiculized through hydrophobic media, such as the lung surfactant (LS). Upon colonization of the lung alveoli, below the LS layer, Streptococcus pneumoniae, causes community-acquired pneumonia, a severe respiratory condition. In this work, we convert PHA NPs into an antimicrobial material by the immobilization of an enzybiotic, an antimicrobial enzyme, via a minimal PHA affinity tag. We first produced the fusion protein M711, comprising the minimized PHA affinity tag, MinP, and the enzybiotic Cpl-711, which specifically targets S. pneumoniae. Then, a PHA nanoparticulate suspension with adequate physicochemical properties for pulmonary delivery was formulated, and NPs were decorated with M711. Finally, we assessed the antipneumococcal activity of the nanosystem against planktonic and biofilm forms of S. pneumoniae. The resulting system displayed sustained antimicrobial activity against both, free and sessile cells, confirming that tag-mediated immobilization of enzybiotics on PHAs is a promising platform for bioactive antimicrobial functionalization.

A study on Sr/Zn phytate complexes: structural properties and antimicrobial synergistic effects against Streptococcus mutans.

Phytic acid (PA) is an abundant natural plant component that exhibits a versatility of applications benefited from its chemical structure, standing out its use as food, packing and dental additive due to its antimicrobial properties. The capacity of PA to chelate ions is also well-established and the formation and thermodynamic properties of different metallic complexes has been described. However, research studies of these compounds in terms of chemistry and biological features are still demanded in order to extend the application scope of PA complexes. The main goal of this paper is to deepen in the knowledge of the bioactive metal complexes chemistry and their bactericide activity, to extend their application in biomaterial science, specifically in oral implantology. Thus, this work presents the synthesis and structural assessment of two metallic phytate complexes bearing the bioactive cations Zn2+ and Sr2+ (ZnPhy and SrPhy respectively), along with studies on the synergic biological properties between PA and cations. Metallic phytates were synthesized in the solid-state by hydrothermal reaction leading to pure solid compounds in high yields. Their molecular formulas were C6H12024P6Sr4·5H2O and C6H12024P6Zn6·6H2O, as determined by ICP and HRES-TGA. The metal coordination bond of the solid complexes was further analysed by EDS, Raman, ATR-FTIR and solid 13C and 31P-NMR spectroscopies. Likewise, we evaluated the in vitro ability of the phytate compounds for inhibiting biofilm production of Streptococcus mutans cultures. Results indicate that all compounds significantly reduced biofilm formation (PA < SrPhy < ZnPhy), and ZnPhy even showed remarkable differences with respect to PA and SrPhy. Analysis of antimicrobial properties shows the first clues of the possible synergic effects created between PA and the corresponding cation in different cell metabolic processes. In overall, findings of this work can contribute to expand the applications of these bioactive metallic complexes in the biotechnological and biomedical fields, and they can be considered for the fabrication of anti-plaque coating systems in the dentistry field.

When microbial biotechnology meets material engineering.

Bacterial biopolymers such as bacterial cellulose (BC), alginate or polyhydroxyalkanotes (PHAs) have aroused the interest of researchers in many fields, for instance biomedicine and packaging, due to their being biodegradable, biocompatible and renewable. Their properties can easily be tuned by means of microbial biotechnology strategies combined with materials science. This provides them with highly diverse properties, conferring them non-native features. Herein we highlight the enormous structural diversity of these macromolecules, how are they produced, as well as their wide range of potential applications in our daily lives. The emergence of new technologies, such as synthetic biology, enables the creation of next-generation-advanced materials presenting smart functional properties, for example the ability to sense and respond to stimuli as well as the capacity for self-repair. All this has given rise to the recent emergence of biohybrid materials, in which a synthetic component is brought to life with living organisms. Two different subfields have recently garnered particular attention: hybrid living materials (HLMs), such as encapsulation or bioprinting, and engineered living materials (ELMs), in which the material is created bottom-up with the use of microbial biotechnology tools. Early studies showed the strong potential of alginate and PHAs as HLMs, whilst BC constituted the most currently promising material for the creation of ELMs.

From Residues to Added-Value Bacterial Biopolymers as Nanomaterials for Biomedical Applications.

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.

Phasin interactome reveals the interplay of PhaF with the polyhydroxyalkanoate transcriptional regulatory protein PhaD in Pseudomonas putida.

Phasin PhaF, a multifunctional protein associated with the surface of polyhydroxyalkanoate (PHA) granules that also interacts with the nucleoid, contributes significantly to PHA biogenesis in pseudomonads. As a protein present on the surface of PHA granules, PhaF participates in granule stabilization and segregation, whereas its deletion has a notable impact on overall transcriptome, PHA accumulation and cell physiology, suggesting more extensive functions besides solely being a granule structural protein. Here, we followed a systematic approach to detect potential interactions of PhaF with other components of the cell, which could pinpoint unexplored functions of PhaF in the regulation of PHA production. We determined the PhaF interactome in Pseudomonas putida KT2440 via pull-down-mass spectrometry (PD-MS) experiments. PhaF complexed with PHA-related proteins, phasin PhaI and the transcriptional regulator PhaD, interactions that were verified to be direct using in vivo two-hybrid analysis. The determination of the PHA granule proteome showed that PhaI and three other potential PhaF interacting partners, but not PhaD, were granule-associated proteins. Analysis of the interaction of PhaF and PhaD with the phaI promoter by EMSA suggested a new role for PhaF in interacting with PhaD and raises new questions on the regulatory system controlling pha gene expression.

Genome sequence and characterization of the bcs clusters for the production of nanocellulose from the low pH resistant strain Komagataeibacter medellinensis ID13488.

Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose-producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT-PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT-PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed.

The TubR-centromere complex adopts a double-ring segrosome structure in Type III partition systems.

In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation. Here, we combine biochemical reconstruction and structural and biophysical analysis to bring light to the architecture of the segrosome complex in Type III partition systems. We present the particular features of the centromere site, tubC, of the model system encoded in Clostridium botulinum prophage c-st. We find that the split centromere site contains two different iterons involved in the binding and spreading of the CBP, TubR. The resulting nucleoprotein complex consists of a novel double-ring structure that covers part of the predicted promoter. Single molecule data provides a mechanism for the formation of the segrosome structure based on DNA bending and unwinding upon TubR binding.

parD toxin-antitoxin system of plasmid R1--basic contributions, biotechnological applications and relationships with closely-related toxin-antitoxin systems.

Toxin-antitoxin systems, as found in bacterial plasmids and their host chromosomes, play a role in the maintenance of genetic information, as well as in the response to stress. We describe the basic biology of the parD/kiskid toxin-antitoxin system of Escherichia coli plasmid R1, with an emphasis on regulation, toxin activity, potential applications in biotechnology and its relationships with related toxin-antitoxin systems. Special reference is given to the ccd toxin-antitoxin system of plasmid F because its toxin shares structural homology with the toxin of the parD system. Inter-relations with related toxin-antitoxin systems present in the E. coli chromosome, such as the parD homologues chpA/mazEF and chpB and the relBE system, are also reviewed. The combined structural and functional information that is now available on all these systems, as well as the ongoing controversy regarding the role of the chromosomal toxin-antitoxin loci, have made this review especially timely.

Repressor CopG prevents access of RNA polymerase to promoter and actively dissociates open complexes.

Replication of the promiscuous plasmid pMV158 requires expression of the initiator repB gene, which is controlled by the repressor CopG. Genes repB and copG are co-transcribed from promoter P(cr). We have studied the interactions between RNA polymerase, CopG and the promoter to elucidate the mechanism of repression by CopG. Complexes formed at 0 degrees C and at 37 degrees C between RNA polymerase and P(cr) differed from each other in stability and in the extent of the DNA contacted. The 37 degrees C complex was very stable (half-life of about 3 h), and shared features with typical open complexes generated at a variety of promoters. CopG protein repressed transcription from P(cr) at two different stages in the process leading to the initiation complex. First, CopG hindered binding of RNA polymerase to the promoter. Second, CopG was able to displace RNA polymerase once the enzyme has formed a stable complex with P(cr). A model for the CopG-mediated disassembly of the stable RNA polymerase-P(cr) promoter complex is presented.

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.