A novel <Emphasis Type="Italic">Geobacteraceae</Emphasis>-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe (III) and Mn (IV) oxides in <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis>

Background  

Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration.     

Establishment and metabolic analysis of a model microbial community for understanding trophic and electron accepting interactions of subsurface anaerobic environments

Background  

Communities of microorganisms control the rates of key biogeochemical cycles, and are important for biotechnology, bioremediation, and industrial microbiological processes. For this reason, we constructed a model microbial community comprised of three species dependent on trophic interactions. The three species microbial community was comprised of Clostridium cellulolyticum, Desulfovibrio vulgaris Hildenborough, and Geobacter sulfurreducens and was grown under continuous culture conditions. Cellobiose served as the carbon and energy source for C. cellulolyticum, whereas D. vulgaris and G. sulfurreducens derived carbon and energy from the metabolic products of cellobiose fermentation and were provided with sulfate and fumarate respectively as electron acceptors.     

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

Background  

The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.     

Characterization of the metabolic shift between oxidative and fermentative growth in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by comparative <Superscript>13</Superscript>C flux analysis

Background  

One of the most fascinating properties of the biotechnologically important organism Saccharomyces cerevisiae is its ability to perform simultaneous respiration and fermentation at high growth rate even under fully aerobic conditions. In the present work, this Crabtree effect called phenomenon was investigated in detail by comparative ¹³C metabolic flux analysis of S. cerevisiae growing under purely oxidative, respiro-fermentative and predominantly fermentative conditions.     

Oxidation of glucose by syntrophic association between <Emphasis Type="Italic">Geobacter</Emphasis> and hydrogenotrophic methanogens in microbial fuel cell

Objective

To investigate a syntrophic interaction between Geobacter sulfurreducens and hydrogenotrophic methanogens in sludge-inoculated microbial fuel cell (MFC) systems running on glucose with an improved electron recovery at the anode.

Results

The presence of archaea in MFC reduces Coulombic efficiency (CE) due to their electron scavenging capability but, here, we demonstrate that a syntrophic interaction can occur between G. sulfurreducens and hydrogenotrophic methanogens via interspecies H₂ transfer with improvement in CE and power density. The addition of the methanogenesis inhibitor, 2-bromoethanesulfonate (BES), resulted in the reduction in power density from 5.29 to 2 W/m³, and then gradually increased to the peak value of 5.5 W/m³ when BES addition was stopped.

Conclusion

Reduction of H₂ partial pressure by archaea is an efficient approach in improving power output in a glucose-fed MFC system using Geobacter sp. as an inoculum.

     

OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis

Background  

The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i) tracer cultivation on ¹³C substrates, (ii) ¹³C labelling analysis by mass spectrometry and (iii) mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis.     

Model-driven elucidation of the inherent capacity of <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> for electricity generation

Background

G. sulfurreducens is one of the commonest microbes used in microbial fuel cells (MFCs) for organic-to-electricity biotransformation. In MFCs based on this microorganism, electrons can be conveyed to the anode via three ways: 1) direct electron transfer (DET) mode, in which electrons of reduced c-type cytochromes in the microbial outer membrane are directly oxidized by the anode; 2) mediated electron transfer (MET) mode, in which the reducing potential available from cell metabolism in the form of NADH is targeted as an electron source for electricity generation with the aid of exogenous mediators; and 3) a putative mixed operation mode involving both electron transfer mechanisms described above (DET and MET). However, the potential of G. sulfurreducens for current output in these three operation modes and the metabolic mechanisms underlying the extraction of the reducing equivalents are still unknown.

Results

In this study, we performed flux balance analysis (FBA) of the genome-scale metabolic network to compute the fundamental metabolic potential of G. sulfurreducens for current output that is compatible with reaction stoichiometry, given a realistic nutrient uptake rate. We also developed a method, flux variability analysis with target flux minimization (FATMIN) to eliminate futile NADH cycles. Our study elucidates the possible metabolic strategies to sustain the NADH for current production under the MET and Mixed modes. The results showed that G. sulfurreducens had a potential to output current at up to 3.710 A/gDW for DET mode, 2.711 A/gDW for MET mode and 3.272 A/gDW for a putative mixed MET and DET mode. Compared with DET, which relies on only one contributing reaction, MET and Mixed mode were more resilient with ten and four reactions respectively for high current production.

Conclusions

The DET mode can achieve a higher maximum limit of the current output than the MET mode, but the MET has an advantage of higher power output and more flexible metabolic choices to sustain the electric current. The MET and DET modes compete with each other for the metabolic resource for the electricity generation.

     

Neuraminidase Inhibitors from marine‐derived actinomycete Streptomyces seoulensis

Aims

This work was performed to characterize new secondary metabolites with neuraminidase (NA) inhibitory activity from marine actinomycete strains.

Methods and Results

An actinomycete strain IFB‐A01, capable of producing new NA inhibitors, was isolated from the gut of shrimp Penasus orientalis and identified as Streptomyces seoulensis according to its 16S rRNA sequence (over 99% homology with that of the standard strain). Repeated chromatography of the methanol extract of the solid‐substrate culture of S. seoulensis IFB‐A01 led to the isolation of streptoseolactone ( 1 ), and limazepines G ( 2 ) and H ( 3 ). The structures of 1 – 3 were determined by a combination of IR, ESI‐MS, 1D (¹H and ¹³C NMR, and DEPT) and 2D NMR experiments (HMQC, HMBC, ¹H‐¹H COSY and NOESY). Compounds 1 – 3 showed significant inhibition on NA in a dose‐dependent manner with IC₅₀ values of 3·92, 7·50 and 7·37 μmol l^?1, respectively.

Conclusions

This is the first report of two new ( 1 and 2 ) and known ( 3 , recovered as a natural product for the first time in the work) NA inhibitors from the marine‐derived actinomycete S. seoulensis IFB‐A01.

Significance and Impact of the Study

The three natural NA inhibitors maybe of value for the development of drug(s) necessitated for the treatment of viral infections.     

An automated growth enclosure for metabolic labeling of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> with <Superscript>13</Superscript>C-carbon dioxide - an <Emphasis Type="Italic">in vivo</Emphasis> labeling system for proteomics and metabolomics research

Background  

Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with ¹³C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate ¹³C from ¹³CO₂ gas. Because of the high cost of ¹³CO₂ it is critical that the design conserve the labeled gas.     

Data from necropsy studies and <Emphasis Type="Italic">in vitro</Emphasis> tissue studies lead to a model for allometric scaling of basal metabolic rate

Background  

The basal metabolic rate (BMR) of a mammal of mass M is commonly described by the power function αM ^β where α and β are constants determined by linear regression of the logarithm of BMR on the logarithm of M (i. e., β is the slope and α is the intercept in regression analysis). Since Kleiber's demonstration that, for 13 measurements of BMR, the logarithm of BMR is closely approximated by a straight line with slope 0.75, it has often been assumed that the value of β is exactly 3/4 (Kleiber's law).     

<Superscript>13</Superscript>C labeling experiments at metabolic nonstationary conditions: An exploratory study

Background  

Stimulus Response Experiments to unravel the regulatory properties of metabolic networks are becoming more and more popular. However, their ability to determine enzyme kinetic parameters has proven to be limited with the presently available data. In metabolic flux analysis, the use of ¹³C labeled substrates together with isotopomer modeling solved the problem of underdetermined networks and increased the accuracy of flux estimations significantly.     

Polar lipid fatty acids,LPS-hydroxy fatty acids,and respiratory quinones of three <Emphasis Type="Italic">Geobacter</Emphasis> strains,and variation with electron acceptor

The polar lipid fatty acids, lipopolysaccharide hydroxy-fatty acids, and respiratory quinones of Geobacter metallireducens str. GS-15, Geobacter sulfurreducens str. PCA, and Geobacter bemidjiensis str. Bem are reported. Also, the lipids of G. metallireducens were compared when grown with Fe³⁺ or nitrate as electron acceptors and G. sulfurreducens with Fe³⁺ or fumarate. In all experiments, the most abundant polar lipid fatty acids were 14:0, i15:0, 16:1ω7c, 16:1ω5c, and 16:0; lipopolysaccharide hydroxy-fatty acids were dominated by 3oh16:0, 3oh14:0, 9oh16:0, and 10oh16:0; and menaquinone-8 was the most abundant respiratory quinone. Some variation in lipid profiles with strain were observed, but not with electron acceptor. D. C. White: Deceased.     

Metabolic engineering for the production of shikimic acid in an evolved <Emphasis Type="Italic">Escherichia coli</Emphasis> strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS^-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG ^fbr, tktA, aroB and aroE, on SA synthesis.     

Cytochromes <Emphasis Type="Italic">c</Emphasis> of the anaerobic methacrylate reducer <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> AM-1

Cytochromes c were found in the cells of the bacterium Geobacter sulfurreducens AM-1 grown on acetate and methacrylate. The periplasmic extract of G. sulfurreducens AM-1 contained about 88% of the total content of cytochromes c of intact cells. The analysis of cytochromes c from the native cells of G. sulfurreducens AM-1, from the periplasmic extract and from the cells treated by an alkaline solution showed the presence of nine proteins containing heme c. The molecular masses of cytochromes c from G. sulfurreducens AM-1 were 12.5, 15.5, 25.7, 29.5, 34.7, 41.7, 50.1, 63.1, and 67.6 kDa; localization of each cytochrome c was determined. Three heme-containing proteins (15.5 kDa, 25.7 kDa, and 29.5 kDa with the most intensive staining) were present mainly in the periplasm of the bacterium. The other two (50.1 and 67.6 kDa) were supposedly localized in the cell membrane. Cytochromes c with the molecular masses of 12.5, 15.5, and 67.6 kDa are considered as possible components of the methacrylate redox system of G. sulfurreducens AM-1.     

Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile

Background

In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated.

Results

The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III).

Conclusion

This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.     

A metabolic signature of long life in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Many Caenorhabditis elegans mutations increase longevity and much evidence suggests that they do so at least partly via changes in metabolism. However, up until now there has been no systematic investigation of how the metabolic networks of long-lived mutants differ from those of normal worms. Metabolomic technologies, that permit the analysis of many untargeted metabolites in parallel, now make this possible. Here we use one of these, ¹H nuclear magnetic resonance spectroscopy, to investigate what makes long-lived worms metabolically distinctive.     

Real‐time PCR for Helicobacter pylori diagnosis. The best tools available

Background

Knowledge of antimicrobial susceptibility, especially to macrolides, has become crucial for the management of Helicobacter pylori infection. Our aim was to evaluate two new PCR kits able to detect H. pylori in gastric biopsies as well as the mutations associated with macrolide resistance.

Materials and Methods

Two hundred successive biopsies (received from gastroenterologists all over France) were used. The two new kits tested were Amplidiag H. pylori+Clari^R from Mobidiag Espoo, Finland, and RIDA^®GENE H. pylori from R‐Biopharm, Darmstadt, Germany. Culture and a validated in‐house real‐time PCR were also performed, and in the case of a positive culture, Etest for clarithromycin was carried out. Discrepancies were solved by looking at the pathologic data.

Results

Culture was positive in 68 cases (34%), and with our in‐house real‐time PCR in these 68 cases plus 5 others (N = 73, 36%). All were also detected by the two new kits. In addition, RIDA^®GENE H. pylori detected one more positive also detected by Amplidiag H. pylori+Clari^R, and Amplidiag detected two other positives. Of these three additional cases, pathology confirmed the positivity for two. Only one case diagnosed by Amplidiag could be considered as a false positive. With regard to clarithromycin resistance, 22 cases were detected. The corresponding mutations (A2142/43G) were all identified with the three PCRs.

Conclusions

These two new kits which have an excellent sensitivity and specificity are convenient to use, adaptable to different thermocyclers, provide quick results, and deserve to be used in H. pylori diagnosis for a better choice of treatment regimen.     

Characterization of proton production and consumption associated with microbial metabolism

Background  

Production or consumption of protons in growth medium during microbial metabolism plays an important role in determining the pH of the environment. Such pH changes resulting from microbial metabolism may influence the geochemical speciation of many elements in subsurface environments. Protons produced or consumed during microbial growth were measured by determining the amount of acid or base added in a 5 L batch bioreactor equipped with pH control for different species including Escherichia coli, Geobacter sulfurreducens, and Geobacter metallireducens.