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.     

Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid: a quantitative multiprobe approach

To better understand how ceramide modulates the biophysical properties of the membrane, the interactions between palmitoyl-ceramide (PCer) and palmitoyl-sphingomyelin (PSM) were studied in the presence of the fluid phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) in membrane model systems. The use of two fluorescent membrane probes distinctly sensitive to lipid phases allowed a thorough biophysical characterization of the ternary system. In these mixtures, PCer recruits POPC and PSM in the fluid phase to form extremely ordered and compact gel domains. Gel domain formation by low PCer mol fraction (up to 12 mol %) is enhanced by physiological PSM levels (approximately 20-30 mol % total lipid). For higher PSM content, a three-phase situation, consisting of fluid (POPC-rich)/gel (PSM-rich)/gel (PCer-rich) coexistence, is clearly shown. To determine the fraction of each phase a quantitative method was developed. This allowed establishing the complete ternary phase diagram, which helps to predict PCer-rich gel domain formation and explains its enhancement through PSM/PCer interactions.     

Backbone assignment of human proliferating cell nuclear antigen

PCNA is an essential factor for DNA replication, repair, chromatin metabolism, and effector of cell-cycle regulatory signals. The assignment of backbone ¹H_N, ¹³C_α, ¹³CO, and ¹⁵N, and sidechain ¹³C_β resonances of the human PCNA homotrimeric ring (∼90 kDa, 261 residues) is reported here.     

Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40 degrees C) inside the coexistence range of liquid-ordered (l(o)) and liquid-disordered (l(d)) phases were investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine(TM)-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The l(o)/l(d) partition coefficients of the probes were determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l(d) phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (<20 nm) of l(o) in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l(d) in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l(o) phase from initially pure l(d), and domain growth being faster in formation of l(d) phase from initially pure l(o).     

Exclusion of a cholesterol analog from the cholesterol-rich phase in model membranes

Vesicles of phosphatidylcholine/cholesterol mixtures show a wide composition range with coexistence of two fluid phases, the 'liquid disordered' (cholesterol-poor) and 'liquid ordered' (cholesterol-rich) phases. These systems have been widely used as models of membranes exhibiting lateral heterogeneity (membrane domains). The distributions of two fluorescent probes (a fluorescent cholesterol analog, NBD-cholesterol, and a lipophilic rhodamine probe, octadecylrhodamine B) in dimyristoylphosphatidylcholine/cholesterol vesicles were studied, at 30 degrees C and 40 degrees C. The steady-state fluorescence intensity of both probes decreases markedly with increasing cholesterol concentration, unlike the fluorescence lifetimes. The liquid ordered to liquid disordered phase partition coefficients K(p) were measured, and values much less than unity were obtained for both probes, pointing to preference for the cholesterol-poor phase. Globally analyzed time-resolved energy transfer results confirmed these findings. It is concluded that, in particular, NBD-cholesterol is not a suitable cholesterol analog and its distribution behavior in phosphatidylcholine/cholesterol bilayers is in fact opposite to that of cholesterol.     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [--> 3)-beta-D-Gal f -(1 --> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [--> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The mannan cores have also been investigated, and are constituted by a (1 --> 6)-alpha-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 --> 2) linked alpha-mannopyranoses.     

Cloning and characterization of the MAL11 gene encoding a high-affinity maltose transporter from Torulaspora delbrueckii

The transport and regulation of maltose utilization by Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species present in home-made corn and rye bread dough, has been investigated. A DNA fragment containing the MAL11 gene from T. delbrueckii (TdMAL11) was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 1884 bp, encoding a 627-amino acid membrane protein, which displays high homology to other yeast maltose transporters. Upstream of TdMAL11, the DNA insert contained a partial ORF (TdMAL12) on the opposite strand, which showed high similarity to the S. cerevisiae MAL12 gene. Sequence analysis, Northern blot and transport measurements indicated that TdMAL11 expression is regulated by the carbon source. Attempts to disrupt TdMAL11 revealed the presence of two functional MAL loci. Disruption of a single copy decreased the V(max) of maltose transport, but not the K(m), whereas the double disruption abolished the uptake of this sugar in T. delbrueckii.     

Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies

Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.