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ADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identified during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specific RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions.

Objective: To analyze the clinical and economic impact of an antimicrobial stewardship program (ASP) targeting urinary tract infections (UTI) caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli., Methods: An observational retrospective study that included adults with a diagnosis of UTI caused by ESBL-producing E. coli admitted to a tertiary care hospital in Barcelona, Spain, between January 2014 and December 2015. The impact of the ASP was analyzed in terms of clinical and economic outcomes., Results: A total of 222 patients met the inclusion criteria and an intervention was made by the ASP team in 104 cases (47%). ASP intervention was an independent variable related to clinical cure (p = 0.008). Other variables influencing clinical outcomes were the McCabe Jackson score (p = 0.005) and outpatient status (p < 0.001). The ASP interventions in this study had no economic impact., Conclusions: Antimicrobial stewardship has a positive clinical impact on UTIs caused by ESBL-producing E. coli. Further prospective studies are needed to assess the economic impact of ASPs on UTI caused by ESBL-producing E. coli., (© The Author 2018. Published by Sociedad Española de Quimioterapia.)

Objective: To analyze the clinical and economic impact of urinary tract infections (UTIs) caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli requiring hospitalization., Methods: Matched cohort study including adults with UTI caused by ESBL-producing E. coli admitted to a tertiary care hospital in Barcelona, Spain, between August 2010 and July 2013. Demographic, clinical and economic data were analyzed., Results: One hundred and twenty episodes of UTI were studied: 60 due to ESBL-producing E. coli and 60 due to non-ESBL-producing E. coli. Bivariate analysis showed that prior antimicrobial treatment (p = 0.007) and ESBL production (p < 0.001) were related to clinical failure during the first 7 days. Multivariate analysis selected ESBL as the sole risk factor for clinical failure (p = 0.002). Regarding the economic impact of infections caused by ESBL-producing E. coli, an ESBL-producing infection cost more than a non-ESBL-producing E. coli infection (mean €4980 vs. €2612). Looking at hospital expenses separately, the total pharmacy costs and antibiotic costs of ESBL infections were considerably higher than for non-ESBL infections (p < 0.001), as was the need for outpatient parenteral antibiotic therapy (OPAT) and its related costs. Multivariate analysis performed for the higher costs of UTI episodes found statistically significant differences for males (p = 0.004), chronic renal failure (p = 0.025), ESBL production (p = 0.008) and OPAT (p = 0.009)., Conclusion: UTIs caused by EBSL-producing E. coli requiring hospital admission are associated with worse clinical and economic outcomes., (Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Production of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria.

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45-85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.

Escherichia coli glycogen metabolism involves the regulation of glgBXCAP operon expression and allosteric control of the GlgC [ADPG (ADP-glucose) pyrophosphorylase]-mediated catalysis of ATP and G1P (glucose-1-phosphate) to ADPG linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC-negative allosteric regulator AMP. The transcriptomic analyses carried out in the present study revealed that, compared with their isogenic BW25113 wild-type strain, glgS-null (ΔglgS) mutants have increased expression of the operons involved in the synthesis of type 1 fimbriae adhesins, flagella and nucleotides. In agreement, ΔglgS cells were hyperflagellated and hyperfimbriated, and displayed elevated swarming motility; these phenotypes all reverted to the wild-type by ectopic glgS expression. Also, ΔglgS cells accumulated high colanic acid content and displayed increased ability to form biofilms on polystyrene surfaces. F-driven conjugation based on large-scale interaction studies of glgS with all the non-essential genes of E. coli showed that deletion of purine biosynthesis genes complement the glycogen-deficient, high motility and high biofilm content phenotypes of ΔglgS cells. Overall the results of the present study indicate that glycogen deficiency in ΔglgS cells can be ascribed to high flagellar propulsion and high exopolysaccharide and purine nucleotides biosynthetic activities competing with GlgC for the same ATP and G1P pools. Supporting this proposal, glycogen-less ΔglgC cells displayed an elevated swarming motility, and accumulated high levels of colanic acid and biofilm. Furthermore, glgC overexpression reverted the glycogen-deficient, high swarming motility, high colanic acid and high biofilm content phenotypes of ΔglgS cells to the wild-type. As on the basis of the present study GlgS has emerged as a major determinant of E. coli surface composition and because its effect on glycogen metabolism appears to be only indirect, we propose to rename it as ScoR (surface composition regulator).

In this work we have investigated the selectivity of lactose permease (LacY) of Escherichia coli (E. coli) for its surrounding phospholipids when reconstituted in binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). Förster resonance energy transfer (FRET) measurements have been performed to investigate the selectivity between a single tryptophan mutant of LacY used as donor (D), and two analogues of POPE and POPG labeled with pyrene in the acyl chains (Pyr-PE and Pyr-PG) used as acceptors. As a difference from previous works, now the donor has been single-W151/C154G/D68C LacY. It has been reported that the replacement of the aspartic acid in position 68 by cysteine inhibits active transport in LacY. The objectives of this work were to elucidate the phospholipid composition of the annular region of this mutant and to determine whether the mutation performed, D68C, induced changes in the protein-lipid selectivity. FRET efficiencies for Pyr-PE were always higher than for Pyr-PG. The values of the probability of each site in the annular ring being occupied by a label (μ) were similar at the studied temperatures (24 °C and 37 °C), suggesting that the lipid environment is not significantly affected when increasing the temperature. By comparing the results with those obtained for single-W151/C154G LacY, we observe that the mutation in the 68 residue indeed changes the selectivity of the protein for the phospholipids. This might be probably due to a change in the conformational dynamics of LacY.

Background: The objective of this study is to analyze the factors that are associated with the adequacy of empirical antibiotic therapy and its impact in mortality in a large cohort of patients with extended-spectrum β-lactamase (ESBL)--producing Escherichia coli and Klebsiella spp. bacteremia., Methods: Cases of ESBL producing Enterobacteriaceae (ESBL-E) bacteremia collected from 2003 through 2008 in 19 hospitals in Spain. Statistical analysis was performed using multivariate logistic regression., Results: We analyzed 387 cases ESBL-E bloodstream infections. The main sources of bacteremia were urinary tract (55.3%), biliary tract (12.7%), intra-abdominal (8.8%) and unknown origin (9.6%). Among all the 387 episodes, E. coli was isolated from blood cultures in 343 and in 45.71% the ESBL-E was multidrug resistant. Empirical antibiotic treatment was adequate in 48.8% of the cases and the in hospital mortality was 20.9%. In a multivariate analysis adequacy was a risk factor for death [adjusted OR (95% CI): 0.39 (0.31-0.97); P = 0.04], but not in patients without severe sepsis or shock. The class of antibiotic used empirically was not associated with prognosis in adequately treated patients., Conclusion: ESBL-E bacteremia has a relatively high mortality that is partly related with a low adequacy of empirical antibiotic treatment. In selected subgroups the relevance of the adequacy of empirical therapy is limited.

We report a thermodynamic study of the effect of calcium on the mixing properties at the air-water interface of two phospholipids that mimic the inner membrane of Escherichia coli: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. In this study, pure POPE and POPG monolayers and three mixed monolayers, χ(POPE) = 0.25, 0.5, and 0.75, were analyzed. We show that for χ(POPE) = 0.75, the values of the Gibbs energy of mixing were negative, which implies attractive interactions. We used atomic force microscopy to study the structural properties of Langmuir-Blodgett monolayers that were transferred onto mica substrate at lateral surface pressures of 25 and 30 mN m(-1). The topographic images of pure POPE and POPG monolayers exhibited two domains of differing size and morphology, showing a step height difference within the range expected for liquid-condensed and liquid-expanded phases. The images captured for χ(POPE) = 0.25 were featureless, and for χ(POPE) = 0.5 small microdomains were observed. The composition that mimics quantitatively the proportions found in the inner membrane of E. coli , χ(POPE) = 0.75, showed large liquid condensed domains in the liquid expanded phase. The extension of each domain was quantitatively analyzed. Because calcium is used in the formation of supported bilayers of negatively charged phospholipids, the possible influence of the nanostructure of the apical on the distal monolayer is discussed.

Although it is generally accepted that Escherichia coli glycogen genes are organized in two tandemly arranged, differentially regulated glgBX and glgCAP operons, RT (reverse transcriptase)-PCR analyses carried out in the present study showed that E. coli cells possess transcripts comprising the five glgBXCAP genes. glg::lacZY expression analyses in cells lacking the region immediately upstream of the glgB gene revealed an almost total abolishment of glgB, glgX and glgC expression, but only a 50-60% reduction of the wild-type glgA and glgP expression levels. Furthermore, similar analyses showed that glgA and glgP expression was almost totally abolished in cells lacking glgA upstream sequences, including glgC, glgB and the asd-glgB intergenic region upstream of glgB. These results indicate that E. coli glgBXCAP genes are organized in a single transcriptional unit controlled by promoter sequences occurring upstream of glgB, and that an alternative suboperonic promoter is located within glgC, driving expression of the glgA and glgP genes. Computer searches for consensus promoters, and analyses of glgB::lacZY and glgA::lacZY expression in cells containing deletions of glgB and glgA upstream sequences identified regions directing glgBXCAP and glgAP expression. 5' RACE (rapid amplification of cDNA ends) analyses located a glgBXCAP transcription start site 155 bp upstream of the glgB initiation codon, and a glgAP transcription start site 359 bp upstream of the glgA initiation codon. Finally, glg::lacZY expression analyses on cells lacking the relA or phoP regulatory genes indicated that both the glgBXCAP operon and the suboperonic promoter driving glgAP expression form part of both the RelA and PhoP-PhoQ regulons.

Using a systematic and comprehensive gene expression library (the ASKA library), we have carried out a genome-wide screening of the genes whose increased plasmid-directed expression affected glycogen metabolism in Escherichia coli. Of the 4123 clones of the collection, 28 displayed a glycogen-excess phenotype, whereas 58 displayed a glycogen-deficient phenotype. The genes whose enhanced expression affected glycogen accumulation were classified into various functional categories including carbon sensing, transport and metabolism, general stress and stringent responses, factors determining intercellular communication, aggregative and social behaviour, nitrogen metabolism and energy status. Noteworthy, one-third of them were genes about which little or nothing is known. We propose an integrated metabolic model wherein E. coli glycogen metabolism is highly interconnected with a wide variety of cellular processes and is tightly adjusted to the nutritional and energetic status of the cell. Furthermore, we provide clues about possible biological roles of genes of still unknown functions.

Biochemical and structural work has revealed the importance of phospholipids in biogenesis, folding and functional modulation of membrane proteins. Therefore, the nature of protein-phospholipid interaction is critical to understand such processes. Here, we have studied the interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) mixtures with the lactose permease (LacY), the sugar/H(+) symporter from Escherichia coli and a well characterized membrane transport protein. FRET measurements between single-W151/C154G LacY reconstituted in a lipid mixture composed of POPE and POPG at different molar ratios and pyrene-labeled PE or PG revealed a different phospholipid distribution between the annular region of LacY and the bulk lipid phase. Results also showed that both PE and PG can be part of the annular region, being PE the predominant when the PE:PG molar ratio mimics the membrane of E. coli. Furthermore, changes in the thermotropic behavior of phospholipids located in this annular region confirm that the interaction between LacY and PE is stronger than that of LacY and PG. Since PE is a proton donor, the results obtained here are discussed in the context of the transport mechanism of LacY., (Copyright 2009 Elsevier B.V. All rights reserved.)

Using the Keio collection of gene-disrupted mutants of Escherichia coli, we have recently carried out a genome-wide screening of the genes affecting glycogen metabolism. Among the mutants identified in the study, Delta mgtA, Delta phoP and Delta phoQ cells, all lacking genes that are induced under low extracellular Mg2+ conditions, displayed glycogen-deficient phenotypes. In this work we show that these mutants accumulated normal glycogen levels when the culture medium was supplemented with submillimolar Mg2+ concentrations. Expression analyses conducted in wild-type, Delta phoP and Delta phoQ cells showed that the glgCAP operon is under PhoP-PhoQ control in the submillimolar Mg2+ concentration range. Subsequent screening of the Keio collection under non-limiting Mg2+ allowed the identification of 183 knock-out mutants with altered glycogen levels. The stringent and general stress responses, end-turnover of tRNA, intracellular AMP levels, and metabolism of amino acids, iron, carbon and sulfur were major determinants of glycogen levels. glgC::lacZY expression analyses using mutants representing different functional categories revealed that the glgCAP operon belongs to the RelA regulon. We propose an integrated metabolic model wherein glycogen metabolism is (a) tightly controlled by the energy and nutritional status of the cell and (b) finely regulated by changes in environmental Mg2+ occurring at the submillimolar concentration range.

ADP sugar pyrophosphatase (AspP) is a member of the 'Nudix' (Nucleoside diphosphate linked to some other moiety X) hydrolase family of enzymes that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) linked to glycogen biosynthesis. In a previous work, we showed that AspP activity is strongly enhanced by both glucose-1,6-bisphosphate and nucleotide-sugars, and by macromolecular crowding. In this work, we show that AspP binds to cell membranes as the bacterial population density increases, c. 30% of the total enzyme remaining membrane associated as glycogen depletes during the stationary phase. This process is not dependent on the stationary transcription factor RpoS, the producer of the bacterial quorum-sensing autoinducer 2 (LuxS), the presence of glycogen granules or glucose availability, but is stimulated by small soluble heat-labile molecule(s) occurring in cell-free spent supernatants of stationary cultures that are acid stabile and base labile. These data further point to AspP as a highly regulated enzyme, and provide a first set of evidences indicating that glycogen metabolism is subjected to regulation by intercellular communication in Escherichia coli.

AC70R1-504 Escherichia coli mutants possess a glgC* gene with a nucleotide change resulting in a premature stop codon that renders a truncated, inactive form of GlgC. Cells over-expressing the wild type glgC, but not those over-expressing the AC70R1-504 glgC*, accumulated high ADPglucose and glycogen levels. AC70R1-504 mutants accumulated glycogen, whereas DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery displayed a glycogen-less phenotype. AC70R1-504 cells with enhanced glycogen synthase activity accumulated high glycogen levels. By contrast, AC70R1-504 cells with high ADPG hydrolase activity accumulated low glycogen. These data further confirm that enterobacteria possess various sources of ADPglucose linked to glycogen biosynthesis.

In this work, using atomic force microscopy (AFM), we have studied the influence of the temperature on the properties of the surface planar bilayers (SPBs) formed with: (i) the total lipid extract of Escherichia coli; (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC) (1:1, mol/mol); and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). According to the height profile analysis we performed, the height of the SPBs of DMPC:POPC were temperature dependent. Separated domains were observed in the SPBs of the POPE:POPG mixture and the E. coli lipid extract. The implication of those domains for the correct insertion of membrane proteins into proteoliposomes is discussed.

The membrane transport protein lactose permease (LacY), a member of the Major Facilitator Superfamily (MFS) containing twelve membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes of: (i) 1,2-dimyristoyl-sn-glycero-3-phosphocoline (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in equimolar proportions; and (ii) Escherichia coli total lipid extract. The structural order of the lipid membranes, in the presence and absence of LacY, was investigated using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) evidenced: (i) the insertion of LacY into the bilayer; and (ii) a surface effect on the membranes. The most dramatic effects were observed when LacY was reconstituted in the E. coli lipid matrix. The effect of the protein on the electrostatic surface potential of each bilayer was also examined using a fluorescent pH indicator, 4-Heptadecyl-7-hydroxycoumarin (HHC). Changes in surface potential were enhanced in the presence of the substrate (i.e. lactose) only when the lipid matrices were charged. These results suggest a role for charged phospholipids (i.e. phosphatidylethanolamine or phosphatidylglycerol) in proton transfer to the amino acids involved in substrate translocation.

Lactose permease (LacY) of Escherichia coli is not only a paradigm for secondary transporters but also for difficulties in two-dimensional (2D) crystallization. In this work we present the progresses achieved in the observation of 2D crystals of wild-type LacY by atomic force microscopy (AFM). Crystals were obtained following reconstitution of LacY in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. Proteolipid sheets (PLSs) 6.4 nm in height were obtained after spreading the samples onto mica. Observations were carried out in liquid medium and in contact mode (CM-AFM). When the crystalline surfaces of the PLSs were imaged regular packing arrangements were observed. The back-Fourier transformation revealed the existence of various orientations mostly consistent with crystals possessing p2 symmetry and unit-cell dimensions: a=13.15 nm, b=16.74 nm, gamma=116 degrees. The characteristics, size, and shape of the repetitive motif could be compatible with dimers of this protein. These preliminary results are compared and discussed with previously reported 2D crystals observed by electron microscopy.

Proteolipid sheets (PLSs) obtained using the vesicle fusion technique on a convenient surface are the base to obtain transmembrane protein biosensors. In this preliminary work, we have screened several physicochemical conditions to optimize the visualization of proteolipid sheets formed between different phospholipid matrices and the membrane protein lactose permease (LacP) by atomic force microscopy (AFM). When LacP was reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, the proteolipid sheets were densely packed with an upper layer that protruded from a background layer. Several lipid protein molar ratios (LPR) were screened. High resolution analysis of the upper layer revealed a quasi-crystalline arrangement formed by small entities that could be attributed to the protein. The approach described here may be suitable for the rational design of biosensors based in other transmembrane proteins.

The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications. [ABSTRACT FROM AUTHOR]

There is an increasing demand on alternatives methods to animal testing. Numerous health parameters have been already studied using in vitro devices able to mimic the essential functions of the organs, being the real-time monitoring and response to stimuli their main limitations. Regarding the health of the gut, the short chain fatty acids, and particularly acetate, have emerged as key biomarkers to evaluate gut healthiness and disease development, although the number of acetate biosensors is still very low. This article presents a microbial biosensor based on fully biocompatible materials which is able to detect acetate in aerobic conditions in the range between 11 and 50 mM, and without compromising the viability and function of either bacteria (>90% viability) or mammalian cells (>80% viability). The detection mechanism is based on the metabolism of acetate by Escherichia coli bacteria immobilized on the transducer surface. Ferricyanide is used as a redox mediator to transfer electrons from the acetate metabolism in the bacterial cells to the transducer. High bacterial concentrations are immobilized in the transducer surface (109 cfu mL−1) by electrodeposition of conductive alginate hydrogels doped with reduced graphene oxide. The results show successful outcomes to exploit bacteria as a biosensing tool, based on the use of inkjet printed transducers, biocompatible materials and cell entrapment technologies. [Display omitted] • Escherichia coli uses acetate as carbon source in aerobic conditions. • Stable, biocompatible, conductive alginate hydrogel is electrodeposited on inkjet printed sensor's working electrode. • Immobilization of high bacterial concentrations of E. coli within alginate hydrogel. • Bacterial metabolism oxidizes acetate, enabling amperometric measurement of electron flow via ferricyanide reduction. • Successful proof-of-concept demonstration of bioelectrochemical acetate detection is performed. [ABSTRACT FROM AUTHOR]

Abstract: In this paper we present a comparative study of supported lipid bilayers (SLBs) and proteolipid sheets (PLSs) obtained from deposition of lactose permease (LacY) of Escherichia coli proteoliposomes in plane. Lipid matrices of two components, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), at a 3:1, mol/mol ratio, were selected to mimic the inner membrane of the bacteria. The aim was to investigate how species of different compactness and stiffness affect the integration, distribution and nanomechanical properties of LacY in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or 1,2-palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). Both compositions displayed phase separation and were investigated by atomic force microscopy (AFM) imaging and force-spectroscopy (FS) mode. PLSs displayed two separated, segregated domains with different features that were characterised by FS and force-volume mode. We correlated the nanomechanical characteristics of solid-like gel phase (L β) and fluid liquid-crystalline phase (L α) with phases emerging in presence of LacY. We observed that for both compositions, the extended PLSs showed a L β apparently formed only by lipids, whilst the second domain was enriched in LacY. The influence of the lipid environment on LacY organisation was studied by performing protein unfolding experiments using the AFM tip. Although the pulling experiments were unspecific, positive events were obtained, indicating the influence of the lipid environment when pulling the protein. A possible influence of the lateral surface pressure on this behaviour is suggested by the higher force required to pull LacY from DPPE:POPG than from POPE:POPG matrices. This is related to higher forces governing protein–lipid interaction in presence of DPPE. [Copyright &y& Elsevier]

Abstract: The phospholipid composition that surrounds a membrane protein is critical to maintain its structural integrity and, consequently, its functional properties. To understand better this in the present work we have performed FRET measurements between the single tryptophan residue of a lactose permease Escherichia coli mutant (single-W151/C154G LacY) and pyrene-labeled phospholipids (Pyr-PE and Pyr-PG) at 37°C. We have reconstituted this LacY mutant in proteoliposomes formed with heteroacid phospholipids, POPE and POPG, and homoacid phospholipids DOPE and DPPE, resembling the same PE/PG proportion found in the E. coli inner membrane (3:1, mol/mol). A theoretical model has been fitted to the experimental data. In the POPE/POPG system, quantitative model calculations show accordance with the experimental values that requires an annular region composed of approximately ∼90mol% PE. The experimental FRET efficiencies for the gel/fluid phase-separated DOPE/POPG system indicate a higher presence of PG in the annular region, from which it can be concluded that LacY shows clear preference for the fluid phase. Similar conclusions are obtained from analysis of excimer-to-monomer (E/M) pyrene ratios. To test the effects of this on cardiolipin (CL) on the annular region, myristoyl-CL and oleoyl-CL were incorporated in the biomimetic POPE/POPG matrix. The experimental FRET efficiency values, slightly larger for Pyr-PE than for Pyr-PG, suggest that CL displaces POPE and, more extensively, POPG from the annular region of LacY. Model fitting indicates that CL enrichment in the annular layer is, in fact, solely produced by replacing PG and that myristoyl-CL is not able to displace PE in the same way that oleoyl-CL does. One of the conclusions of this work is the fact that LacY inserts preferentially in fluid phases of membranes. [ABSTRACT FROM AUTHOR]

Abstract: We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli (E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids. [Copyright &y& Elsevier]

Phosphatidylethanolamine (PE) and phosphatidylgycerol (PG) are the main components of the inner membrane of Escherichia coli. Mixtures of PE and PG mimicking the proportions found in E. coli have been extensively used to reconstitute transmembrane proteins as lactose permease (LacY) in proteoliposomes because in this environment the protein shows maximal activity. Hence, the study of the physicochemical properties of this phospholipid matrix becomes of potential interest. In previous studies, , we used atomic force microscopy (AFM) and force spectroscopy (FS) to study the topographic and nanomechanical properties of supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and of POPE and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). The study reported here was extended for completeness to asymmetric SLBs obtained by the Langmuir−Blodgett (LB) method. Thus, we prepared SLBs with the proximal leaflet extracted at 30 mN·m−1 and the distal leaflet extracted at 25 mN·m−1. We prepared SLBs with both leaflets with same composition (POPG/POPG), and also with the proximal leaflet of POPE and the distal leaflet of POPG or POPE:POPG (3:1, mol/mol). The topography of the SLBs acquired in liquid was compared with the topography of the monolayers acquired in air. Breakthrough (Fy) and adhesion forces (Fadh) of SLBs were extracted from force curves. The values obtained are discussed in terms of the possible involvement of the nanomechanical properties of the SLBs in membrane protein insertion. The results provide means for the observation that insertion of LacY in POPE:POPG (3:1, mol/mol) occurs preferentially in the fluid phase, which is the phase with the lower Fy and the higher Fadh. [ABSTRACT FROM AUTHOR]

Abstract: Atomic force microscopy (AFM) was used to study the influence of a membrane protein, lactose permease of Escherichia coli (LacY), on the surface spreading behavior and the features of self-assembled phospholipids bilayers on mica. The miscibility of phospholipids used, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was investigated by surface pressure area isotherm measurements at the air–water interface. A composition with an equimolar proportion of POPC and DMPC was used to form the liposomes. Surface layers formed with DMPC:POPC (0.5:0.5, mol/mol) or LacY reconstituted in proteoliposomes with the same phospholipid composition were imaged by using AFM. When lactose permease was reconstituted in DMPC:POPC (0.5:0.5, mol/mol), self-assembled structures that remained firmly adsorbed onto the mica surface were observed. These sheets had an irregular shape and their upper layer was more corrugated than that obtained for the phospholipid matrix. [Copyright &y& Elsevier]

In this work the porin Omp1 of Serratia marcescens was expressed in a porin deficient mutant (Escherichia coli UH302) and its functionality studied following the accumulation of ciprofloxacin in bacteria. The protein was extracted, purified and reconstituted in proteoliposomes of different composition (lipopolysaccharide (LPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)). Maximum extraction of the detergent was achieved applying different steps of dialysis and centrifugation. Proteolipid sheets with different composition were spread onto mica and observed by atomic force microscopy. Two-dimensional crystal of Omp1 was not observed in any case due to low resolution achieved. Judging from the images features POPC is the most suitable phospholipid to enhance 2D lattice formation for Omp1. [Copyright &y& Elsevier]